Expansion of human mesenchymal stem/stromal cells on temporary liquid microcarriers

BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION: The prospect of a temporary microcarrier that can be used to expand cells and then ‘disappear’ for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality

Effects of Very Low Nicotine Content Cigarettes on Smoking Behavior and Biomarkers of Exposure in Menthol and Non-menthol Smokers.

IntroductionBecause 30% of cigarettes sold in the United States are characterized as menthol cigarettes, it is important to understand how menthol preference may affect the impact of a nicotine reduction policy.MethodsIn a recent trial, non-treatment-seeking smokers were randomly assigned to receive very low nicotine cigarettes (VLNC; 0.4 mg nicotine/g tobacco) or normal nicotine cigarettes (NNC; 15.5 mg/g) for 20 weeks. On the basis of preference, participants received menthol or non-menthol cigarettes. We conducted multivariable regression analyses to examine whether menthol preference moderated the effects of nicotine content on cigarettes per day (CPD), breath carbon monoxide (CO), urinary total nicotine equivalents (TNE), urinary 2-cyanoethylmercapturic acid (CEMA), and abstinence.ResultsAt baseline, menthol smokers (n = 346) reported smoking fewer CPD (14.9 vs. 19.2) and had lower TNE (52.8 vs. 71.6 nmol/mg) and CO (17.7 vs. 20.5 ppm) levels than non-menthol smokers (n = 406; ps < .05). At week 20, significant interactions indicated that menthol smokers had smaller treatment effects than non-menthol smokers for CPD (-6.4 vs. -9.3), TNE (ratio of geometric means, 0.22 vs. 0.10) and CEMA (ratio, 0.56 vs. 0.37; ps < .05), and trended toward a smaller treatment effect for CO (-4.5 vs. -7.3 ppm; p = .06). Odds ratios for abstinence at week 20 were 1.88 (95% confidence interval [CI] = 0.8 to 4.4) for menthol and 9.11 (95% CI = 3.3 to 25.2) for non-menthol VLNC smokers (p = .02) relative to the NNC condition.ConclusionsAlthough menthol smokers experienced reductions in smoking, toxicant exposure, and increases in quitting when using VLNC cigarettes, the magnitude of change was smaller than that observed for non-menthol smokers.ImplicationsResults of this analysis suggest that smokers of menthol cigarettes may respond to a nicotine reduction policy with smaller reductions in smoking rates and toxicant exposure than would smokers of non-menthol cigarettes

Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain

AbstractWith the ever-increasing clinical application of cell-based therapies, it is considered critical to develop systems that facilitate the storage and distribution of cell therapy products (CTPs) between sites of manufacture and the clinic. For such systems to be realized, it is essential that downstream bioprocessing strategies be established that are scalable, reproducible and do not influence the viability or function of the living biologic. To this end, we examined alginate-encapsulation as a method to heighten the preservation of human adipose-derived stem cells (hASCs) during hypothermic storage, and establish a scalable process for high-volume production. A drop-wise method for scalable alginate bead generation, using calcium as the cross-linker, was modified to enable the yield of up to 3500 gelled beads per minute. The effect of alginate concentration on the viscosity of non-gelled sodium alginate and the mechanical properties and internal structure of calcium-crosslinked alginate in response to different alginate and calcium concentrations were investigated. Mechanical strength was chiefly dependent on alginate concentration and 1.2% alginate cross-linked with 100mM calcium chloride could withstand stress in the order of 35kPa. Upon selection of appropriate parameters, we demonstrated the suitability of using this method for immobilizing human stem cells. Encapsulated hASCs demonstrated no loss in cell viability, and had a uniform distribution after high-volume production. Following storage, released cells were able to attach and recover a normal morphology upon return to culture conditions. Thus we present a scalable method for stem cell encapsulation and storage for application within the cell therapy supply chain

The effect of scale and interfacial tension on liquid–liquid dispersion in in-line Silverson rotor–stator mixers

AbstractThe effect of scale, processing conditions, interfacial tension and viscosity of the dispersed phase on power draw and drop size distributions in three in-line Silverson rotor–stator mixers was investigated with the aim to determine the most appropriate scaling up parameter. The largest mixer was a factory scale device, whilst the smallest was a laboratory scale mixer. All the mixers were geometrically similar and were fitted with double rotors and standard double emulsor stators. 1wt.% silicone oils with viscosities of 9.4mPas and 339mPas in aqueous solutions of surfactant or ethanol were emulsified in single and multiple pass modes. The effect of rotor speed, flow rate, dispersed phase viscosity, interfacial tension and scale on drop size distributions was investigated.It was found that for all three scales, power draw is the sum of the rotor and flow contributions, with proportionality constants, PoZ and k1, that are practically scale independent. Sauter mean drop size appeared to correlate better with tip speed than energy dissipation rate. For ethanol/water solutions, mean drop size correlated well with Weber number based on interfacial tension, but for surfactant solutions effective interfacial tension gave better correlations with Weber number

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase

Continued primer synthesis at stalled replication forks contributes to checkpoint activation

An increased number of primer–template junctions generated by PCNA, Pol-δ, and Pol-ε at stalled replication forks activates Chk1

Latent class analysis of sexual health markers among men and women participating in a British probability sample survey.

BACKGROUND: Despite known associations between different aspects of sexual health, it is not clear how patterning of adverse sexual health varies across the general population. A better understanding should contribute towards more effective problem identification, prevention and treatment. We sought to identify different clusters of sexual health markers in a general population, along with their socio-demographic, health and lifestyle correlates. METHODS: Data came from men (N = 5113) and women (N = 7019) aged 16-74 who reported partnered sexual activity in the past year in Britain's third National Survey of Sexual Attitudes and Lifestyles, undertaken in 2010-2012. Latent class analysis used 18 self-reported variables relating to adverse sexual health outcomes (STI and unplanned pregnancy, non-volitional sex, and sexual function problems). Correlates included socio-demographics, early debut, alcohol/drug use, depression, and satisfaction/distress with sex life. RESULTS: Four classes were found for men (labelled Good Sexual Health 83%, Wary Risk-takers 4%, Unwary Risk-takers 4%, Sexual Function Problems 9%); six for women (Good Sexual Health 52%, Wary Risk-takers 2%, Unwary Risk-takers 7%, Low Interest 29%, Sexual Function Problems 7%, Highly Vulnerable 2%). Regardless of gender, Unwary Risk-takers reported lower STI/HIV risk perception and more condomless sex than Wary Risk-takers, but both were more likely to report STI diagnosis than Good Sexual Health classes. Highly Vulnerable women reported abortion, STIs and functional problems, and more sexual coercion than other women. Distinct socio-demographic profiles differentiated higher-risk classes from Good Sexual Health classes, with depression, alcohol/drug use, and early sexual debut widely-shared correlates of higher-risk classes. Females in higher-risk classes, and men with functional problems, evaluated their sex lives more negatively than those with Good Sexual Health. CONCLUSIONS: A greater prevalence and diversity of poor sexual health appears to exist among women than men in Britain, with more consistent effects on women's subjective sexual well-being. Shared health and lifestyle characteristics of higher-risk groups suggest widespread benefits of upstream interventions. Several groups could benefit from tailored interventions: men and women who underestimate their STI/HIV risk exposure, women distressed by low interest in sex, and women experiencing multiple adverse outcomes. Distinctive socio-demographic profiles should assist with identification and targeting