Recovery Of Viable Cells Research Articles

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

BackgroundThe role of CD4+ regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs. MethodsHIV-uninfected (HIV−) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b+ and CD39+ Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. ResultsPercentages of CD120b+ Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4h of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. ConclusionThe data suggest that percentages of CD120b+ Tregs and CD39+ Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV− and HIV+ men. However, for measurement of CD120b+ Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells.

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 10(6) cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state.

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).

In vitro probiotic characterization of Lactobacillus strains from fermented radish and their anti-adherence activity against enteric pathogens.

In this study, we evaluated the probiotic properties of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus fermentum strains isolated from fermented radish. All the strains survived the simulated oro-gastrointestinal transit condition and showed significantly higher adherence to Caco-2 cells compared with the probiotic strain Lactobacillus rhamnosus GG. The strains showed broad-spectrum antimicrobial activity, autoaggregation, and coaggregation capacity with pathogens. Furthermore, the Lactobacillus strains inhibited the adherence of Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Salmonella choleraesuis to the Caco-2 cell line. The strains possessed bile salt hydrolase activity and their cholesterol-lowering activity in vitro was above 50% in the presence of bile. Strains of L. plantarum and L. pentosus possessed the plantaricin-encoding plnEF gene. In addition, the Lactobacillus strains maintained about 80% cell viability after freeze-drying in the presence of a combination of 5% skim milk and 5% maltodextrin as cryoprotectant, and 70% recovery of cell viability was observed in the absence of any cryoprotectant.

Impact of Detachment Methods on M2 Macrophage Phenotype and Function

The methods of cell detachment influence phenotype and function of human macrophages cultured in vitro. However, comparative studies defining the influence of cell detachment techniques on secondary characterization of M1 or M2 polarized macrophages are largely absent from the literature. In this study we evaluated the impact of trypsin, accutase, EDTA, PBS, and cell scraping on: A. cell recovery, B. phenotype and C. function of in vitro polarized macrophages. Our data demonstrate that while exposure to trypsin or accutase yields highly efficient recovery of viable cells, such chemical cleavage results in loss of select M2 cell surface markers with correlative changes in cell function. In contrast, phenotype and function are maintained following detachment by EDTA on ice. Our data suggest that seemingly “trivial” changes in methodologies for macrophage detachment induce both variable and profound changes on cell phenotype and function which can dramatically impact the results of polarization experiments.

The photodynamic antibacterial effects of silicon phthalocyanine (Pc) 4.

The emergence of antibiotic-resistant strains in facultative anaerobic Gram-positive coccal bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), is a global health issue. Typically, MRSA strains are found associated with institutions like hospitals but recent data suggest that they are becoming more prevalent in community-acquired infections. It is thought that the incidence and prevalence of bacterial infections will continue to increase as (a) more frequent use of broad-spectrum antibiotics and immunosuppressive medications; (b) increased number of invasive medical procedures; and (c) higher incidence of neutropenia and HIV infections. Therefore, more optimal treatments, such as photodynamic therapy (PDT), are warranted. PDT requires the interaction of light, a photosensitizing agent, and molecular oxygen to induce cytotoxic effects. In this study, we investigated the efficacy and characterized the mechanism of cytotoxicity induced by photodynamic therapy sensitized by silicon phthalocyanine (Pc) 4 on (a) methicillin-sensitive Staphylococcus aureus (MSSA) (ATCC 25923); (b) community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) (ATCC 43300); and (c) hospital acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) (PFGE type 300). Our data include confocal image analysis, which confirmed that Pc 4 is taken up by all S. aureus strains, and viable cell recovery assay, which showed that concentrations as low as 1.0 μM Pc 4 incubated for 3 h at 37 °C followed by light at 2.0 J/cm2 can reduce cell survival by 2–5 logs. These results are encouraging, but before PDT can be utilized as an alternative treatment for eradicating resistant strains, we must first characterize the mechanism of cell death that Pc 4-based PDT employs in eliminating these pathogens.

C-2012: Post-thaw characterization of cord blood-derived mesenchymal stromal cells cryopreserved for up to five years

Mesenchymal stromal cells (MSCs) are increasingly used as therapeutic agents for a variety of diseases. Because long-term banking of MSCs is critical for their clinical applications, we sought to determine whether the viability, proliferation potential, capacity for differentiation, migration ability and amenability for genetic manipulation are maintained in MSCs cryopreserved for up to five years. MSCs were established from the mononuclear cell fraction of an umbilical cord blood (CB) sample. Primary adherent MSC monolayers were sub-cultured until passage 12 and aliquots of various cell passages were frozen at −70 °C in the presence of 10% dimethylsulfoxide (Me 2 SO) and 10% fetal bovine serum. MSCs were thawed rapidly at 37 °C, sub-cultured to about 80% confluency, trypsinized, and subjected to trypan blue viability test, seeded in the presence of osteogenic and chondrogenic media, analyzed by flow cytometry for expression of surface markers and by RT-PCR for gene expression of tissue markers, and assessed for migration response. We found that although the viability decreased by up to 20% post-thaw, the proliferation potential of CB-MSCs as measured by population doubling level of 7 was not affected by cryopreservation, nor was their ability to differentiate to osteoblasts (by staining of calcium deposits using Alizarin red) and to chondrocytes (by staining of proteoglycans using Alcian blue). The MSCs retained positive expression for the stromal markers CD73, CD90 and CD105 and negative expression for the hematopoietic markers CD34 and CD45. In addition, they expressed pluripotency genes and early tissue markers for muscle, bone and cartilage. Furthermore, the MSCs retained their migratory response towards the chemokine stromal cell-derived factor, which is up-regulated at sites of tissue injury. In conclusion, CB-derived MSCs cryopreserved for up to 5 years at −70 °C in the presence of 10% Me 2 SO demonstrate high-efficiency recovery of viable, proliferative and functional cells which can be employed for clinical transplantation or manipulated for gene therapy.

C-41: Pre-cryopreservation processing and storage: A biologist’s perspective on the balance between manufacturing efficiency and post-thaw quality

The ex vivo storage of cells at low temperatures for extended periods of time has been one of the most important advances that led to the creation of biobanks and the widespread therapeutic use of cellular products. Most cellular products used today spend a large portion of their product lifecycle at hypothermic temperatures or cryopreserved and stored frozen in freezers. As it is generally understood in the biobanking industry that the length and temperature of pre-cryopreservation storage can influence the post-thaw recovery of cells, biobankers have empirically established thresholds for the “optimal” conditions for the: shipment of raw material to centralized manufacturing sites, cell selection, donor testing and processing. Unfortunately, there is a general lack of appreciation of the impact that pre-cryopreservation processing and storage has on critical cell properties that are crucial to post-thaw recovery of viable cells. This presentation will provide a biologist’s perspective on the impact that cell manufacturing conditions can have on the quality of cellular therapeutics. Emerging data on the hypothermic storage lesion and cryoinjury of red blood cells and hematopoietic progenitor cells will be discussed with a focus on changes to cell metabolism, oxidative injury and membrane remodeling. The impact of the biological changes on the ability for cells to respond to the physical and chemical stresses encountered during cryopreservation will be introduced. Current quality control methods and international regulations will be briefly described. Recent clinical data on the effects of hypothermic storage and cryopreservation on recipient outcomes will be reviewed. While there is a practical / commercial need to optimize the pre-cryopreservation manufacturing efficiency of cellular products, consideration must be given to the impact and dependency that each manufacturing step has on the quality and potency of the final product.

Cytoprotective effect of short-term pretreatment with proanthocyanidin on human gingival fibroblasts exposed to harsh environmental conditions.

Our previous study showed that exposing mouse fibroblasts to proanthocyanidin (PA) for only 1 min accelerated cell proliferation in a concentration-dependent manner. In this study, exposing human gingival fibroblasts (HGFs) to PA for 1 min similarly accelerated the proliferative response of the cells. Besides the accelerated proliferative response, PA showed a cytoprotective effect on HGFs exposed to harsh environmental conditions; short-term exposure of HGFs in the mitotic phase to pure water or physiological saline resulted in a lower recovery of viable cells. Pretreatment and concomitant treatment with PA improved the low recovery of cells exposed to pure water or physiological saline. In addition, HGFs exposed to PA for 1 min proliferated well even after being cultured in serum-free medium. In 100% confluent HGFs, being cultured in serum-free medium resulted in a high intracellular reactive oxygen species (ROS) level, but pretreatment with PA prevented the cells from increasing intracellular ROS. Thus, the results suggest that a short-term PA treatment exerts cytoprotective effects on HGFs exposed to harsh environmental conditions by improving the intracellular oxidative stress response.

A scale down process for the development of large volume cryopreservation

The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48h or 72h in recovery cultures.Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.

Human-derived raw materials: Controlled, consistent collections enable successful manufacturing of cell-based regenerative medicine products (TECH1P.847)

Abstract Human cells are critical raw materials for manufacturing cell therapy products, but introduce significant variability. HemaCare controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 86,799 cellular apheresis collections in the last 7 years for research, clinical trials, and commercial products. HemaCare’s unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.66% MNC±7.1% (mean±1 SD). RBC contamination was low, with hematocrit averaging 1.78%±0.7%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times. HemaCare’s laboratory is equipped with Miltenyi Biotec technology for isolation of raw material into purified cellular subpopulations. The consistency and viability of the purified end products are measured with flow cytometry. Using BioLife Solutions’ serum-free and protein-free cGMP CryoStor™ cryopreservation media with purified cells, post-thaw recovery rates of cell fractions have been above 95%; dendritic cell and macrophage have demonstrated recovery rates of ≥90%. CryoStor™, in combination with freezing in the BioCision CoolCell™ freezing container, has enabled HemaCare to standardize the cryopreservation process, reducing variability while optimizing post-thaw viable cell recovery of its research products.

Development of stabilizers for live cell preservation at room temperature (779.10)

Cyropreservation at ultra‐low temperatures utilizing freezing mediums containing DMSO is the current best practice for long term storage of mammalian cells. Such conditions can impart devastating losses caused by common failures of the cold storage systems as well as the sensitivity of valuable cell types both primary and engineered to the freezing and thawing process. Hence recovery of viable cells from the frozen state is challenging and as is typical for many cell types only a fraction of the cells survive with the fully desired phenotype. Based on the studies of extremophiles such as tardigrades, rotifers or brine shrimp that can survive for many years in the dry state we hypothesized that techniques and protocols could be developed that mimic this molecular phenomena in eukaryotic cell cultures. Many previous candidate approaches or the last 15 years have failed to preserve cells under these conditions over the short term and no method to date has been successful at preserving cells under such conditions for more than a few hours. We applied a combinatorial screening approach to identify formulations containing both biostabilizers that preserve air‐dried eukaryotic cells at ambient temperature. The use specific biostabilizers in specific formulations protect cells from the stress of dehydration, dry‐storage, and rehydration while preventing the execution of apoptosis programs. We report here that we are able to achieve statistically significant improvements in cell survival rates and preservation time as compared to conventionally published dry down methods. These results provide a roadmap for achieving room temperature storage of mammalian cells in a dry state. The technology can be further developed for products that extend cell survival at ambient temperatures. This technology could eventually be applied to ambient storage of eukaryotic cell stocks for biomedical research, toxicology screens, cell‐based therapeutics and blood supply logistics.

Single-use centrifugation solution for volume reduction and cell washing process in cell therapy manufacturing

Current bioprocessing technologies have limited applications in cell therapy manufacturing as they are optimized for the manufacture of recombinant proteins. For example, most cell harvest technologies are efficient in removing cells but not in maximizing viable cell density, viability, washing, and recovery; parameters that are most important for cell therapy manufacturing. Most commonly cells are retained by either centrifugation or filtration based devices. These approaches are often problematic. Conventional centrifugation based devices cause stress and nutrient deprivation to the cells in the pellet which results in low viability, while filtration based devices often suffer from issues such as clogging, retention of cell debris, and shear stress on cells. We have developed the first single-use fluidized-bed centrifugation systems that are completely closed. Our results show that kSep systems can concentrate diverse types of cells with high recovery while maintaining high viability. Additionally, kSep systems can remove cell debris, light particulate impurities, all while significantly reducing aggregation of cells. We have designed these systems without any rotary seals (providing reduced sterility risk) or filters (for reduced issues from clogging). Once captured and concentrated, the cells can efficiently be washed, manipulated, and harvested. This technology is a breakthrough for applications requiring maintenance of cellular integrity during processing. Results show >90% recovery of cells with unchanged viability and 99.9% removal of contaminants. Our automated systems are currently being used for cell therapy, recombinant proteins, and vaccine manufacturing processes.

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.

Delayed Hematopoietic Recovery After Autologous Stem Cell Transplantation In Patients Receiving Arsenic Trioxide-Based Therapy For Acute Promyelocytic Leukemia

Introduction Arsenic trioxide (ATO)-based therapy for acute promyelocytic leukemia (APL) is highly effective and has been incorporated into standard therapeutic algorithms for patients with newly diagnosed and relapsed disease. Therapy for patients with high-risk or relapsed APL also includes high-dose chemotherapy with autologous stem cell transplantation (autoSCT). Both treatments have independently been demonstrated to improve long-term disease-free survival; however, the use of ATO prior to autoSCT has not been well studied. We sought to determine whether use of ATO prior to autologous stem cell collection impacts hematopoietic recovery following autoSCT. Methods Clinical histories of 31 consecutive patients undergoing autoSCT for APL at our institution since 1994 were reviewed. Viable CD34-positive cell recovery from cryo-preserved autograft products was assessed by pre- and post-thaw 7-amino-actinomycin D (7-AAD) staining. Student's t-tests and chi-squared tests were used for statistical analyses. Results A total of 32 autoSCTs were performed in this population. Twenty-four transplants (75.0%) were performed for patients in first complete remission (CR1) versus 8 transplants (25.0%) for patients in second complete remission (CR2). Seven of the 32 transplants (21.8%) were performed for patients who received ATO therapy prior to autologous stem cell collection. One patient received ATO with initial induction and consolidation therapy, 1 patient received ATO with initial consolidation therapy only, and 5 patients received ATO with re-induction therapy for relapsed disease. A significantly higher proportion of patients who received ATO were transplanted in CR2 (71.5% vs 12.0%, p<0.01). There were otherwise no significant differences in clinical characteristics or risk features between the ATO pre-treated and the ATO-naïve transplant recipients, including: median age at diagnosis (45 years vs 44 years, p=0.62), median age at transplant (46 years vs 45 years, p=0.36), white blood cell count >10,000/uL at presentation (28.6% vs 45.8%, p=0.42), mean CD34-positive cells collected (94.5 x106/kg vs 69.9x106/kg, p=0.35), or mean CD34-positive cells infused (41.4x106/kg vs 39.7x106/kg, p=0.90). Four cases (12.5%) of delayed hematopoietic recovery were identified, defined as requiring >16 days after stem cell infusion to recover an absolute neutrophil count (ANC) >1,000/uL. Three of 7 patients (43%) who received prior ATO had delayed hematopoietic recovery, compared with 1 of 24 patients (4.2%) not previously treated with ATO (p<0.01). Compared to ATO-naïve patients, ATO-treated patients experienced significantly longer time to ANC recovery (mean 19.7 days vs 10.4 days, p<0.01; median 14 days vs 10 days). In addition, ATO-treated patients required significantly longer transplant hospitalizations (37.2 days vs 24.6 days, p<0.01). Platelet recovery, defined as a sustained platelet count >50,000/uL, was also significantly delayed in the ATO-treated group (198.8 days vs 36.5 days, p<0.01; median 81 vs 18 days). The only significant predictor of delayed hematopoietic recovery was prior treatment with ATO (75.0% vs 14.2%, p<0.01). Of 7 assessable APL patient samples, the mean viable CD34-positive cell recovery was 39 +/- 25.1%. This was significantly lower than the viable CD34-recovery (71.3 +/- 27.3%, p=0.02) evaluated in a random selection of 11 other cryo-preserved autologous stem cell products from patients with lymphoma (n=2) and multiple myeloma (n=9). Samples from 2 ATO-treated APL patients who experienced delayed hematopoietic recovery exhibited viable CD34-positive cell recoveries of only 3% and 26%. Conclusions There is an association between ATO exposure and delayed hematopoietic recovery after autoSCT for APL, but a causal relationship is not yet established. Because of the increasing frequency with which ATO is being used in the front-line treatment of APL, larger studies are warranted to validate these findings and to determine the potential mechanism of delayed hematopoietic recovery after autoSCT. Lower-than-expected viable CD34-positive cell recovery from APL patients suggests a possible detrimental contribution of prior ATO exposure. Our institution is now prospectively examining pre- and post-thaw CD34-positive cell recovery, viability, and colony-forming assays prior to autoSCT in patients who have previously received ATO. Disclosures: No relevant conflicts of interest to declare.

Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 μm constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 10(2) CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

New decontamination method based on caprylic acid in combination with citric acid or vanillin for eliminating Cronobacter sakazakii and Salmonella enterica serovar Typhimurium in reconstituted infant formula

The antimicrobial effects of natural compounds (caprylic acid, CA; citric acid, CTA; and vanillin, VNL) on the inactivation of Cronobacter sakazakii and Salmonella enterica serovar Typhimurium were examined in reconstituted infant formula. The samples were treated with: 1) CA, CTA, or VNL alone (0, 10, 20, 30, 40, 60, and 80mM); 2) a combination of CA (10 and 20mM) and CTA (15 and 30mM); and 3) a combination of CA (10 and 20mM) and VNL (15 and 30mM), at mild feeding temperatures (40°C and 45°C), and the bacterial populations were assayed periodically (0, 5, 10, and 30min). For both bacteria, the combined treatments had marked synergistic antimicrobial effects compared with the sum of the effects of each individual treatment. For example, there was no noticeable reduction (P>0.05) in the population of C. sakazakii following an individual treatment with 20mM CA or 30mM CTA for 5min at 40°C, whereas the population was reduced to undetectable levels (reduction>7.3 log CFU/ml) following treatment with a combination of CA and CTA (20 CA+30 CTA for 5min at 40°C). As the temperature increased, the bactericidal effect was stronger at all time points with a synergistic effect. In a validation assay using a low level inoculum (approximately 103CFU/ml) of desiccation-stressed bacteria in certain conditions, the combined treatments (e.g., CA 10mM+CTA 30mM for 5min at 45°C for C. sakazakii, and CA 10mM+VNL 15mM for 10min at 45°C for S. Typhimurium) completely destroyed the bacteria with no recovery of cell viability. Disintegration of the membrane and changes in the cell structure or morphology, such as plasmolysis and membrane disruption, were detected by flow cytometry and electron microscopy, respectively. These methods use antimicrobials that could be applied as food additives in infant formula, which may help to eliminate bacteria.

A Novel Approach for Assured Sterility of Medical Devices

A Novel Approach for Assured Sterility of Medical Devices

Multiparametric evaluation of the cytoprotective effect of the Mangifera indica L. stem bark extract and mangiferin in HepG2 cells

Mango (Mangifera indica L.) stem bark extract (MSBE) is a natural product with biological properties and mangiferin is the major component. This paper reported the evaluation of the protective effects of MSBE and mangiferin against the toxicity induced in HepG2 cells by tert-butyl hydroperoxide or amiodarone. Nuclear morphology, cell viability, intracellular calcium concentration and reactive oxygen species (ROS) production were measured by using a high-content screening multiparametric assay. MSBE and mangiferin produced no toxicity below 500 mg/ml doses. A marked recovery in cell viability, which was reduced by the toxicants, was observed in cells pre-exposed to MSBE or mangiferin at 5-100 mg/ml doses. We also explored the possible interaction of both products over P-glycoprotein (P-gp). MSBE and mangiferin above 100 mg/ml inhibited the activity of P-gp in HepG2 cells. MSBE and mangiferin showed cytoprotective effects of against oxidative damage and mitochondrial toxicity induced by xenobiotics to human hepatic cells but it seemed that other constituents of the extract could contribute to MSBE protective properties. In addition, the drug efflux should be taken into account because of the inhibition of the P-gp function observed in those cells exposed to both natural products.

Photoprotective effect of Undaria crenata against ultraviolet B-induced damage to keratinocytes

Chronic exposure of the skin to ultraviolet B (UVB) radiation induces oxidative stress, which plays a crucial role in the induction of skin cancer. The brown alga Undaria crenata is a potential source of antioxidant and anti-apoptotic compounds due to its capacity to produce protective compounds against environmental factors, including UV radiation. The aim of this study was to investigate the photoprotective properties of an U. crenata ethanol extract (UCE) against UVB-induced cell damage in human HaCaT keratinocytes. UCE exhibited absorbing effect of UVB (280-320nm) and scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species induced by hydrogen peroxide and UVB rays. Furthermore, electron spin resonance spectrometry revealed the significant scavenging effect of UCE against superoxide anion and hydroxyl radical. UCE reduced UVB-induced apoptosis, as shown by a decrease in apoptotic bodies and nuclear and DNA fragmentation, resulting in the recovery of cell viability. UCE also decreased the degree of UVB-induced oxidative stress to lipids, proteins, and DNA as shown by a decrease in 8-isoprostane level, protein carbonylation and DNA tails. These results suggest that UCE protects human keratinocytes against UVB-induced oxidative stress.