Retention Of Cell Viability Research Articles

Co-encapsulation of probiotics with acylglycerols in gelatin-gum arabic complex coacervates: Stability evaluation under adverse conditions

Co-encapsulation of acylglycerols and probiotics may improve the resistance of probiotics to adverse conditions. In this study, three probiotic microcapsule models were constructed using gelatin (GE)-gum arabic (GA) complex coacervate as wall material: microcapsules containing only probiotics (GE-GA), microcapsules containing triacylglycerol (TAG) oil and probiotics (GE-T-GA) and microcapsules containing diacylglycerol (DAG) oil and probiotics (GE-D-GA). The protective effects of three microcapsules on probiotic cells under environmental stresses (freeze-drying, heat treatment, simulated digestive fluid and storage) were evaluated. The results of cell membrane fatty acid composition and Fourier transform infrared (FTIR) spectroscopy revealed that GE-D-GA could improve the fluidity of cell membrane, maintain the stability of protein and nucleic acid structure, and decrease the damage of cell membrane. These characteristics supported the high freeze-dried survival rate (96.24 %) of GE-D-GA. Furthermore, regardless of thermotolerance or storage, GE-D-GA showed the best cell viability retention. More importantly, GE-D-GA provided the best protection for probiotics under simulated gastrointestinal conditions, as the presence of DAG reduced cell damage during freeze-drying and the degree of contact between probiotics and digestive fluids. Therefore, co-microencapsulation of DAG oil and probiotics is a promising strategy to resist adverse conditions.

MRNA Delivery to T Cells with Retention of Cell Viability, Proliferation and Functionality Using a Novel Vector-Free Intracellular Delivery Method ‘Soluporation‘

Membrane-disruption-based methods that enable intracellular delivery of various cargo types for clinical applications have been proposed as attractive candidates as next-generation delivery modalities because of potential benefits including safety, regulation and production [1]. Electroporation is the most widely used such method, but while it can be efficient for delivery of nucleic acids to some cell types, toxicity can be high, particularly in primary cells. We recently reported a novel vector-free technology, Solupore™ that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying compositions, properties and sizes [2]. A permeabilizing delivery solution that contains a low level of ethanol is used as the permeabilizing agent. The technology achieves delivery and reversal of cell permeabilization by precisely controlling the contact of the target cells with this solution. The procedure is rapid and is completed in less than 5 min. We have demonstrated that delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner and we have termed the method ‘soluporation’.In this study, we evaluated the ability of Solupore™ to deliver mRNA to primary human T cells and compared this with electroporation. GFP mRNA was delivered to T cells. GFP expression and cell viability were measured at 24 hr post-transfection. Cell proliferation was assessed at days 1-7 using an automated cell counter and functionality was examined by measuring IFNγ secretion.GFP expression in ‘soluporated’ cells ranged between 30-40% while expression in electroporated cells was variable and ranged between 60 and 98%. The viability of soluporated cells was similar to control untreated cells whereas viability in electroporated cells was reduced by 40-50%. The proliferation of soluporated cells over 7 days following transfection was similar to control cells while the proliferation of electroporated cells was significantly reduced. IFNγ secretion by soluporated cells was also unchanged compared to control while that of electroporated cells was significantly reduced.This study demonstrates that Solupore™ can achieve mRNA delivery to T cells with retention of cell viability and retention of proliferation and IFNγ capacity. The technology is gentle yet highly reproducible, compatible with high throughput and automated cell processing and has the potential to enable a broad range of T cell engineering applications.

Evaluation of cell compatibility of surfaces patterned with hydroxyapatite nanopowders on Ti surfaces

Hydroxyapatite (HA) is a widely used biomaterial for coatings on prostheses, which is used to repair, replace and restore the function of traumatized or diseased bone. In the present study, template-assisted electrohydrodynamic atomization (TAEA) was used to pattern Ti surfaces (convex, concave and flat) with different HA nanoparticles, in order to evaluate the cellular responses. A comparison of biocompatibility between surfaces coated with locally prepared (SL) and commercially available HA nanopowders was carried out. The cytotoxicity and cellular responses to these substrates (i.e., cell adhesion, morphology and proliferation) were evaluated using human osteoblast-like cells (HOS) in vitro. The results demonstrate that Ti surfaces coated with HA (SL) and commercial HA supported HOS cell attachment and proliferation without eliciting any deleterious substances. Moreover, retention of cell viability and morphology of osteoblast-like cells were observed once cells were in contact with the surfaces and were comparable to that of the tissue culture control. Interestingly, cells attached to HA (SL) elicited a better response. Proliferation of cells on convex surfaces was relatively higher in comparison to that of the concave and flat surfaces, which could be due to increased surface area of the HA (SL).

Advances in Viable Ice-free Cryopreservation of Heart Valves

Investigation of heart valve cryopreservation has been employed as a model for development of new methods of tissue preservation based upon vitrification and nanowarming using Fe nanoparticles. Cryoprotectant cytotoxicity can be reduced by performing the last cryoprotectant/nanoparticle exposure step below zero degrees centigrade at -10C. Tissue viability outcomes can be improved by supplementation of cryoprotectant formulations with disaccharides and nanowarming can rewarm such complex tissues with retention of cell viability from storage temperatures below -135oC to -25oC in 80-100 seconds. It is anticipated that ice-free tissue cryopreservation methods for tissues up to 50 mLs can be developed that do not require the use of nanowarming, since we are already close to achieving this with heart valves at 30 mL volumes. However, at larger volumes nanowarming will likely continue to be the best warming method for retention of tissue cell viability. Further studies to optimize cryopreservation of cardiac muscle, the somewhat fibrous muscle band at the base of heart valves, and pulmonary and aortic arteries need to be performed since it is clear that different heart valve components vary in their preservation requirements. It is anticipated that other complex tissues may also have components with different cryopreservation requirements including nanowarming.

Vitrification of Heart Valve Tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80°C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80°C with long-term storage preferred at or below -135°C.

Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis.

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y.pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y.pestis at 37°C.

Conductive hydro drying through refractance window drying – An alternative technique for drying of Lactobacillus plantarum (NCIM 2083)

The process of drying improves the shelf-life of a range of food products. Considering drying of probiotics, retention of cell viability is challenging, yet crucial. The impact of various drying process on probiotics viability is well documented. This study was aimed to prepare probiotic powders of Lactobacillus plantarum with a suitable combination of prebiotics by refractance window (RW) drying, a variant of the conductive hydro drying technique. Properties of RW dried synbiotic powder were investigated and compared with freeze dried samples. RW dried synbiotic powder showed lesser moisture content, better flowability, and porous flaky structures in its surface morphology. RW dried synbiotics showed acceptable probiotic viability and stability under simulated oral-gastrointestinal conditions. The findings of this study highlight a new approach for commercial production of probiotic powders.

LB50 - Efficient vector-free engineering of MSC with retention of cell viability and differentiation potential

LB50 - Efficient vector-free engineering of MSC with retention of cell viability and differentiation potential

Development and Evaluation of Bioformulations Using Enrichment Material from Plant Growth Promoting Fluorescent Pseudomonads

Increase in soil fertility, plant growth promotion and suppression of phytopathogens are the targets of the bioformulation industry that leads to the development of ecofriendly environment. The aim of this study is to evaluate different enrichment materials for the development of effective bioformulation with prolonged shelf life. Twentysix fluorescent pseudomonad isolates were screened for antagonistic activity against Fusarium oxysporum. Selected isolate TS2 identified as Pseudomonas sp. on the bases of internal transcribed spacer (ITS) sequence analysis showed production of indole-3-acetic acid (IAA), siderophore, ammonia, cellulase, phenazine antibiotics and phosphate solubilisation. Skimmed milk, sucrose, molasses and trehalose were used as enrichment material for development of talc-based bioformulation of TS2. Sucrose-amended bioformulation followed by molasses was found to be the best among developed bioformulations for supporting TS2 population. However, in presence of pathogen, bioformulation enriched with molasses showed maximum enhancement in dry weight of chickpea taken as test crop. The finding of this study suggests that enrichment of carrier permits the retention of cell viability thus increasing the effectiveness of the active material.

Polysaccharide Multilayer Nanoencapsulation of Insulin-Producing β-Cells Grown as Pseudoislets for Potential Cellular Delivery of Insulin

This paper describes the use of a layer-by-layer nanocoating technique for the encapsulation of insulin-producing pancreatic beta-cell spheroids (pseudoislets) within chitosan/alginate multilayers. We used pseudoislets self-organized from a population of the insulinoma cell line MIN6, derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta-cells, as an experimental model for the study of cell nanoencapsulation. The maintenance of spheroid morphology and retention of cell viability and metabolic functionality was demonstrated postencapsulation. By depositing an additional protein-repelling phosphorylcholine-modified chondroitin-4-sulfate layer, the coatings were found to shield effectively access of large molecules of the immune systems to the antigen-presenting cell surfaces. Transmission electron microscopy analysis of the encapsulated pseudoislets revealed that the coating did not damage the cell structure. In addition, nanoencapsulation permits the cells to respond to changes in extracellular glucose and other insulin secretagogues by releasing insulin with a profile similar to that of nonencapsulated cells. These results suggest that this nanofilm encapsulation technique has the characteristics required for the efficient transplantation of cellular engineered beta-cells as a cell replacement therapy for type 1 diabetes. This encapsulation method is general in scope and has implications for use in a variety of cellular therapeutics employing engineered tissues from cells generated in vitro from various sources, including those using genetic and cellular engineering techniques.

Effect of disaccharides on survival during storage of freeze dried probiotics

The aim of this study was to investigate the effects of protective media and different relative vapour pressures (RVPs) on the survival of probiotics during freeze drying and subsequent storage, to determine the optimal conditions for the production of freeze dried probiotics at industrial scale, ensuring a high survival rate. The effect of protective media i.e. reconstituted skimmed milk (RSM) or either of the cryoprotective disaccharides lactose, trehalose, sucrose, maltose, lactose + maltose and lactose + trehalose on the survival of a probiotic culture was assessed at five different RVP environments (0.0%, 11.4%, 33.2%, 44.1% and 76.1%) at room temperature in freeze-dried systems. RVP was shown to have a significant effect on the survival rates of the probiotic cultures Lactobacillus paracasei NFBC 338 and Lactobacillus rhamnosus GG following freeze drying in RSM. Interestingly, retention of cell viability was greatest for cells stored at 11.4% RVP, but was compromised at all other RVPs tested. However, an increased tolerance to freeze drying was observed for L. rhamnosus GG when dried in the presence of disaccharides in the order of trehalose = lactose + maltose ⩾ lactose + trehalose ⩾ maltose > lactose > sucrose. Survival studies over a 38–40 day storage period indicated that trehalose and lactose + maltose were the most effective cryoprotective additives, especially notable at 0.0 and 11.4% RVP. At all other RVPs tested, viability was compromised. Crystallisation of the disaccharides was observed to be a detrimental factor affecting the survival of Lactobacillus during storage at high RVP, where an inverse relationship was shown to exist between the % RVP and the glass transition temperature (Tg) of the disaccharides.

UV-A mediated induction of carotenoid accumulation in Dunaliella bardawil with retention of cell viability

The effect of adding UV-A radiation (320-400 nm) to photosynthetically active radiation (PAR, 400-700 nm) during growth of the photosynthetic marine microalga Dunaliella bardawil was investigated in this work in terms of cell growth and carotenoid production. Although signs of slow cell growth (slight reduction of chlorophyll and protein content) were observed after 24 h of cell exposure to UV-A (40 micromol photons m(-2) s(-1) and 70 micromol photons m(-2) s(-1)) plus 140 micromol photons m(-2) s(-1) PAR , 84 h exposure to these UV-A conditions slightly stimulated cell growth and increased the photosynthetic efficiency of the exposed cultures. The enhanced cell growth was coupled with an increase in total carotenoid content. Besides beta-carotene as the major pigment, increases in the well-known antioxidants lutein and zeaxanthin of about 3-fold and 5-fold, respectively, were determined in cultures exposed to UV-A radiation of 70 micromol photons m(-2) s(-1)for 84 h. As a consequence, far from being negative to cell growth, low and medium UV-A radiation are stress factors that could be successfully applied to long-term processes for large scale carotenoid production using D. bardawil cultures with retention of cell viability. UV-A exposure has the advantage of being a factor either easily applied or removed as required, in contrast to other nutrient stresses, which require medium replacement for their application.

Discovery and development of biological agents to control crop pests

Thousands of potential microbial biocontrol agents have been isolated from agricultural fields and crops during research over the last 80 years, yet only a few are in commercial use. Recently, public health and safety concerns about the environmental impact of chemical pesticides have led to consideration of biological control as a natural approach to maintaining crop health. Despite environmental incentives and strong research efforts, commercialization of biocontrol agents has been slow to evolve. The momentum of the chemical industry is difficult to shift, and fermentation processes tend to be more expensive to operate than synthetic chemical processes.Yet there is a demand for biological control products, especially in agricultural niche markets, where there is no chemical competitor. However, given this market demand, the fundamental methods of economical large-scale production and application of biological control agents are lacking. Many aspects of biocontrol agent production and development represent untrodden territory in the progression of industrial fermentation technology beyond its well-established food and pharmaceuticals niche. Distinguishing them from traditional fermentation products, biocontrol agents must not only be produced in high yield but must also meet the following quality criteria: high (near 100%) retention of cell viability with maintenance of crop compatibility and consistent bioefficacy during several months of storage. Research examples will be reviewed to illustrate the challenges and strategies of developing processes to manufacture and deliver biological agents for insect, weed, and plant disease control.

Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes.

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to < 1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained > 90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na(+)-free buffer at pH 7.4, pHi did not rise and cell viability remained > 80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5-1 microM) at pH 7.4, the MPT was prevented and cell viability remained > 80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30-40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.

DNA and RNA Synthesis by Postpubertal Undescended Testis In Vitro

To study the spermatogenic potential of postpubertal undescended testis, the 3H-thymidine and 14C-uridine incorporations into normally descended and inguinal undescended testes were examined. The results suggest that DNA synthesis by the inguinal undescended testis is remarkably inhibited but, on the contrary, RNA synthesis is not. The cell-proliferative ability of postpubertal undescended testis may be impaired in spite of the retention of cell viability in the testis.

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40°C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.

Cell-surface modification with an iodinatible imidoester to enhance radiolabeling

A procedure to enhance incorporation of radioactive iodine into cell-surface proteins is described. The effects of chemical modification of cells with the iodinatible haptens N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) and methyl- p-hydroxybenzimidate HCl (Wood reagent) are compared. The Wood reagent, that does not alter the charge of the modified amino group, is superior in retention of cell viability and function. Cell-surface modification with the Wood reagent prior to radiolabeling is particularly useful for detecting antigens not readily iodinated using standard techniques.

Immobilized cells

Cells of microbes and of higher organisms may be immobilized by a number of methods for a variety of purposes. Although the study of cell immobilization is comparatively novel, the methods that have been developed are very effective and there are few indications that further, greatly superior techniques are likely to evolve. Cells are best immobilized by aggregation, by adsorption onto a support material or by entrapment within gels, of which the natural polysaccharides K-carrageenan and calcium alginate have proved the most useful. Aggregation of cells usually involves heat treatment or chemical cross-linkage and causes total loss of cell viability. It is most suitable for the immobilization of cells so as to contain a single enzymic activity: many of the commercial preparations of glucose isomerase consist of aggregated cells. Adsorption of cells to surfaces is a gentle and simple technique: efficient immobilization is aided by the correct choice of pore size in the support material. Cell viabilities and activities are retained but adsorbed cells may be removed from supports fairly readily. Entrapment within gels allows the retention of cell viability and activity and by supplying full growth media, cells can be made to multiply within the beads of gel, giving very high cell densities. Polyacrylamide has been used satisfactorily for cell entrapment but has been superseded in industrial processes by K-carrageenan or calcium alginate. Industrial processes known to employ immobilized cells include the production of L-malic acid and L-aspartic acid and various steroid conversions. Very many other processes using immobilized cells have been studied at the academic level. Immobilization in polysaccharide gels, in particular, sometimes gives unexpected properties of longevity or activity to cells. These phenomena are largely unexplained but may be due to the gel material’s influencing the chemical composition of the immediate environment of the cells. The use of immobilized cells has not yet made a large impact on the fermentation industries but these are early days in the development of new technology and it is to be expected that processes involving immobilized cells will find wider industrial use in the near future.

Macrophage-mediated cytostasis of neoplastic hemopoietic cells: Cytofluorometric analysis of the reversible cell cycle block

Nonactivated mouse peritoneal macrophages inhibit the proliferation of neoplastic hemopoietic cells in vitro. This effect is dependent upon the number of adherent macrophages present in cultures of hemopoietic tumor cells and can be documented by various parameters used as indices of cell proliferation. The two-layer soft agar culture system permits analysis of the regulatory functions of macrophage-derived diffusible substances under conditions of extremely low cell density and where macrophage-tumor cell contact is prevented by the gel matrix. The ability of the underlayers of macrophages to inhibit colony formation by hemopoietic tumor cells indicates that such an effect can be mediated by factor(s) elaborated by macrophages. Evidence that the mode of action of macrophages on tumor cell proliferation is cytostatic rather than cytotoxic is the capability of the macrophages to retard tumor cell growth in a particular phase of the cell cycle with retention of cell viability. Growth inhibition could be demonstrated to be reversible, with tumor cells entering normal cell cycle distributions shortly after being removed from macrophages or macrophage-derived factors. Cytofluorometric analysis of cell cycle inhibition correlated with viable cell counts and mitotic indices and confirms the suitability of this method for studying tumor cell proliferation.