Highest Cell Survival Research Articles

Optimization of cryoprotectant formulation to enhance the viability of Lactiplantibacillus plantarum NBC99 isolated from human origin

Freeze drying has been well applied in the preparation of high-efficiency viability probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which poses a problem for further application. In this study, various kinds of cryoprotectant formulations (skim milk, maltodextrin, and sucrose) were tested to enhance the survival of Lactiplantibacillus plantarum NBC99 after freezing and freeze-drying. An I-optimal experimental design-oriented optimization model was presented to optimize the cryoprotective medium, and the highest cell survival was observed with 25% skim milk, 8.71% maltodextrin, and 1.13% sucrose cryoprotectant as the optimum condition. L. plantarum NBC99 has been a good potential strain for the manufacture of an industrial probiotic, and this research has aimed to investigate the long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria. The results showed the potential value of freeze-dried probiotic L. plantarum NBC99 culture for commercialization.

Optimization of cryoprotectant formulation to enhance the viability of Lactobacillus brevis ED25: Determination of storage stability and acidification kinetics in sourdough

In this study, various kinds of cryoprotectant (skim milk, lactose, and sucrose) formulations were tested to enhance the survival of Lactobacillus brevis ED25 after freezing and freeze-drying. A Box–Behnken experimental design was used to optimize cryoprotective medium and the highest cell survival was observed with the 17.28% skim milk, 2.12% lactose, and 10% sucrose cryoprotectant as the optimum condition. The structural and physicochemical characteristics of freeze-dried powder were acceptable for application with regards to particle surface morphology, moisture and water activity (Aw), glass transition temperature (Tg), Fourier transform infrared spectra, X-ray structure, and also storage stability under the refrigeration and room temperature conditions. Accelerated storage test based on Arrhenius equation could be used to predict the freeze-dried bacterial shelf life but only with a certain degree of predictability for long-term storage. The acidification kinetics of fresh and stored culture in sourdough fermentation was also described on the basis of the Gompertz equation. Practical applications Freezing and storage are crucial factors for the viability and acidification power of starter culture. Therefore, various types and concentrations of cryoprotectants have been used to preserve the microorganisms. L. brevis ED25 has been a good potential for the manufacture of industrial sourdoughs and this research has aimed to investigate long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria and also determine the acidification power in sourdough. The results showed the potential value of freeze-dried L. brevis ED25 culture for commercialization.

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

A nonconventional method for drying of Pseudomonas aeruginosa and its comparison with conventional methods

In this study, preparation of dried cultures of Pseudomonas aeruginosa using nonconventional drying method, namely, low-temperature low-humidity (LTLH) drying was investigated. The effect of carrier materials (whey protein, corn starch, and trehalose) was examined one at a time and also in combinations (to explore the synergistic effect). The results were compared with those obtained using spray drying and freeze drying in terms of cell survival and dry cell powder yield. The powder samples were analyzed also for morphology, flowability, particle size, and moisture content. In LTLH drying, good cell survival was observed along with high powder yield when compared with that in spray drying. Corn starch showed the highest cell survival (91%) and powder yield (94%, w/w) among the carrier materials employed besides resulting in good cell survival (65%) even after a storage period of 6 months.

Effect of Lycium barbarum polysaccharide on expression of vascular endothelial growth factor and hypoxia inducible factor-1α in ultraviolet B-radiated HaCaT cells

Objective To evaluate effects of Lycium barbarum polysaccharide (LBP) on expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in ultraviolet B (UVB) -radiated HaCaT cells. Methods Conventionally cultured HaCaT cells were divided into control group and LBP groups, which were firstly treated with DMEM, 12.5, 25.0, 50.0 and 100 μg/ml LBP solution respectively for 4 hours, and then were irradiated by UVB at different intensity of 0, 20, 40, 60 mJ/cm2 separately. After 24-hour continuing culture, CCK-8 assay was performed to determine the cell survival rate, and an enzymatic-biochemical method to estimate the activity of superoxide dismutase (SOD) . RT-PCR and Western blot analysis were conducted to measure the mRNA and protein expression of HIF-1α and VEGF respectively. Results Compared with the control group at the same UVB radiation dose, the 12.5-, 25.0-and 100.0-μg/ml LBP groups showed different extents of increase in survival rates of UVB-radiated cells (P < 0.05) , and the 50.0-μg/ml LBP group showed the highest cell survival rate (P < 0.01) . Among all the LBP groups, SOD activity was highest in the 50.0-μg/ml LBP group (P < 0.01) . Along with the increase of UVB radiation dose, the mRNA and protein expression of HIF-1α and VEGF all gradually increased. Compared with the control group, the 50.0-μg/ml LBP group could effectively reduce the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells (all P < 0.05) . Conclusion LBP may play a role in protecting cells from UVB radiation-mediated damage, likely by influencing the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells. Key words: Ultraviolet rays; Lycium barbarum; Polysaccharides; Endothelial growth factors; Hypoxia inducible factor 1α; HaCaT cells

Synthesis of Injectable Alginate Hydrogels with Muscle-Derived Stem Cells for Potential Myocardial Infarction Repair

Myocardial infarction (MI), caused by the occlusion of the left ventricular coronary artery, leads to the loss of cardiomyocytes and, potentially, heart failure. Cardiomyocytes in adult mammals proliferate at an extremely low rate and thus, a major challenge in MI treatment is supplementing exogenous cells and keeping them viable in MI areas. To address this challenge, injecting hydrogels encapsulating cells into MI areas, to compensate for the loss of cardiomyocytes, shows promise. This study synthesized two types of alginate hydrogels, based on self-crosslinking (SCL) and calcium ion crosslinking (Ca2+) in varying formulations. The hydrogels encapsulated living muscle-derived stem cells (MDSCs) and their performance was evaluated in terms of optimizing cell viability during the injection process, as well as the live/dead rate after long-term cultivation. The morphology of the hydrogel-encapsulated cells was characterized by scanning electronic microscopy (SEM) and live/dead cells were examined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining) assay. The mechanical properties of the hydrogels were also determined via a rheometer, to identify their influence on cell viability during the injection process and with respect to long-term cultivation. The SCL hydrogel with a 0.8% alginate and 20% gelatin formulation resulted in the highest cell viability during the injection process, and the Ca2+ hydrogel composed of 1.1% alginate and 20% gelatin maintained the highest cell survival rate after two months in culture.

Adjusting the Chemical and Physical Properties of Hydrogels Leads to Improved Stem Cell Survival and Tissue Ingrowth in Spinal Cord Injury Reconstruction: A Comparative Study of Four Methacrylate Hydrogels

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A suitable combination of modern hydrogel materials, modified to effectively bridge the lesion cavity, combined with appropriate stem cell therapy seems to be a promising approach to repair spinal cord damage. We demonstrate the synergic effect of porosity and surface modification of hydrogels on mesenchymal stem cell (MSC) adhesiveness in vitro and their in vivo survival in an experimental model of SCI. MSCs were seeded on four different hydrogels: hydroxypropylmethacrylate-RGD prepared by heterophase separation (HPMA-HS-RGD) and three other hydrogels polymerized in the presence of a solid porogen: HPMA-SP, HPMA-SP-RGD, and hydroxy ethyl methacrylate [2-(methacryloyloxy)ethyl] trimethylammonium chloride (HEMA-MOETACl). Their adhesion capability and cell survival were evaluated at 1, 7, and 14 days after the seeding of MSCs on the hydrogel scaffolds. The cell-polymer scaffolds were then implanted into hemisected rat spinal cord, and MSC survival in vivo and the ingrowth of endogenous tissue elements were evaluated 1 month after implantation. In vitro data demonstrated that HEMA-MOETACl and HPMA-SP-RGD hydrogels were superior in the number of cells attached. In vivo, the highest cell survival was found in the HEMA-MOETACl hydrogels; however, only a small ingrowth of blood vessels and axons was observed. Both HPMA-SP and HPMA-SP-RGD hydrogels showed better survival of MSCs compared with the HPMA-HS-RGD hydrogel. The RGD sequence attached to both types of HPMA hydrogels significantly influenced the number of blood vessels inside the implanted hydrogels. Further, the porous structure of HPMA-SP hydrogels promoted a statistically significant greater ingrowth of axons and less connective tissue elements into the implant. Our results demonstrate that the physical and chemical properties of the HPMA-SP-RGD hydrogel show the best combination for bridging a spinal cord lesion, while the HEMA-MOETACl hydrogel serves as the best carrier of MSCs.

Investigation of the factors influencing the survival of Bifidobacterium longum in model acidic solutions and fruit juices

The survival of Bifidobacterium longum NCIMB 8809 was studied during refrigerated storage for 6weeks in model solutions, based on which a mathematical model was constructed describing cell survival as a function of pH, citric acid, protein and dietary fibre. A Central Composite Design (CCD) was developed studying the influence of four factors at three levels, i.e., pH (3.2–4), citric acid (2–15g/l), protein (0–10g/l), and dietary fibre (0–8g/l). In total, 31 experimental runs were carried out. Analysis of variance (ANOVA) of the regression model demonstrated that the model fitted well the data. From the regression coefficients it was deduced that all four factors had a statistically significant (P<0.05) negative effect on the log decrease [log10N0 week−log10N6 week], with the pH and citric acid being the most influential ones. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate and strawberry. The highest cell survival (less than 0.4log decrease) after 6weeks of storage was observed in orange and pineapple, both of which had a pH of about 3.8. Although the pH of grapefruit and blackcurrant was similar (pH ∼3.2), the log decrease of the former was ∼0.5log, whereas of the latter was ∼0.7log. One reason for this could be the fact that grapefruit contained a high amount of citric acid (15.3g/l). The log decrease in pomegranate and strawberry juices was extremely high (∼8logs). The mathematical model was able to predict adequately the cell survival in orange, grapefruit, blackcurrant, and pineapple juices. However, the model failed to predict the cell survival in pomegranate and strawberry, most likely due to the very high levels of phenolic compounds in these two juices.

Disruption of the gene encoding glutamate dehydrogenase affects growth, amino acids catabolism and survival of Lactobacillus plantarum UC1001

The role played by glutamate dehydrogenase (GDH) in Lactobacillus plantarum was investigated by preparing a GDH-deficient mutant UC1001G. The growth rate and acidification displayed by this mutant were lower than those of the wild-type UC1001. Compared with UC1001G, UC1001 increased the synthesis of proteins that confer a competitive advantage of cell resistance. When both strains were used as adjunct starter for cheese-making, the highest cell survival was found for UC1001. Cheeses made with the adjunct of UC1001 showed the lowest level of free amino acids and the highest content of volatile organic compounds. Both strains produced high level of γ-aminobutyric acid in cheese and under simulated gastrointestinal conditions. The findings of this study provide an evidence of the important role of GDH on growth and survival of Lb. plantarum under different environmental conditions that influences some important functional features of the strain.