Understanding cell culture dynamics: a tool for defining protocol parameters for improved processes and efficient manufacturing using human embryonic stem cells.

Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm2, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour-1 ± 1.5x10-3 over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive).

Effect of in vitro selenium supplementation on sperm quality in asthenoteratozoospermic men.

Sperm DNA damage, excessive oxidative stress and decrease in motility may lead to low fertilisation or poor assisted reproductive techniques outcomes in asthenoteratozoospermic men. Selenium was considered as essential element for male reproductive functions. Selenium has important role in enzymatic process for elimination of excessive reactive oxygen species and helps to maintain membrane integrity. The aim of this study was to determine the effect of selenium supplementation on sperm quality, DNA fragmentation, mitochondrial membrane potential and membrane lipid peroxidation during sperm sampling in vitro at different times. In this experimental study, semen samples were collected from 50 asthenoteratozoospermic men. Samples were divided into two groups as control group and test group (incubated with 2 µg/ml selenium at 37°C for 2, 4 and 6 hr). Motility and viability were assessed based on WHO 2010 criteria. Mitochondrial membrane potential, sperm DNA fragmentation and malondialdehyde levels were evaluated in each group. Results revealed that motility, viability and mitochondrial membrane potential were significantly higher in the test group (p < .05). Also malondialdehyde levels were significantly lower in the test group (p < .03). DNA fragmentation significantly decreased in the test group after 6 hr of incubation (p < .02). In conclusion, in vitro selenium supplementation may protect spermatozoa from maltreatment effect of reactive oxygen species (ROS) during sperm sampling via keeping enzymatic and antioxidant process in optimum condition.

Combined numerical and experimental biomechanical characterization of soft collagen hydrogel substrate.

This work presents a combined experimental-numerical framework for the biomechanical characterization of highly hydrated collagen hydrogels, namely with 0.20, 0.30 and 0.40% (by weight) of collagen concentration. Collagen is the most abundant protein in the extracellular matrix of animals and humans. Its intrinsic biocompatibility makes collagen a promising substrate for embedding cells within a highly hydrated environment mimicking natural soft tissues. Cell behaviour is greatly influenced by the mechanical properties of the surrounding matrix, but the biomechanical characterization of collagen hydrogels has been challenging up to now, since they present non-linear poro-viscoelastic properties. Combining the stiffness outcomes from rheological experiments with relevant literature data on collagen permeability, poroelastic finite element (FE) models were developed. Comparison between experimental confined compression tests available in the literature and analogous FE stress relaxation curves showed a close agreement throughout the tests. This framework allowed establishing that the dynamic shear modulus of the collagen hydrogels is between 0.0097 ± 0.018 kPa for the 0.20% concentration and 0.0601 ± 0.044 kPa for the 0.40% concentration. The Poisson's ratio values for such conditions lie within the range of 0.495-0.485 for 0.20% and 0.480-0.470 for 0.40%, respectively, showing that rheology is sensitive enough to detect these small changes in collagen concentration and thus allowing to link rheology results with the confined compression tests. In conclusion, this integrated approach allows for accurate constitutive modelling of collagen hydrogels. This framework sets the grounds for the characterization of related hydrogels and to the use of this collagen parameterization in more complex multiscale models.

Does sputum culture affect the management and/or outcome of community-acquired pneumonia?

This prospective study was conducted in 6 hospitals in Edmonton, Canada to determine the factors associated with obtaining sputum for culture and the effect of sputum culture on the management of patients with community-acquired pneumonia (CAP). Participants were 1362 adults who were hospitalized with CAP. Sputum was obtained from 539 (39.6%) patients, of which 507 (94.1%) were good quality, acceptable for culture; 171 (33.7%) of these had a positive sputum culture. Levofloxacin, cefuroxime and azithromycin were the most common antibiotics prescribed for the groups with positive sputum culture and no sputum collection. Positive sputum culture was demonstrated in only a small number of patients with CAP; this did not affect antimicrobial therapy or mortality.

Does sputum culture affect the management and/or outcome of community-acquired pneumonia?

This prospective study was conducted in 6 hospitals in Edmonton, Canada to determine the factors associated with obtaining sputum for culture and the effect of sputum culture on the management of patients with community-acquired pneumonia [CAP]. Participants were 1362 adults who were hospitalized with CAP. Sputum was obtained from 539 [39.6%] patients, of which 507 [94.1%] were good quality, acceptable for culture; 171 [33.7%] of these had a positive sputum culture.Levofloxacin, cefuroxime and azithromycin were the most common antibiotics prescribed for the groups with positive sputum culture and no sputum collection. Positive sputum culture was demonstrated in only a small number of patients with CAP; this did not affect antimicrobial therapy or mortality

Community-acquired pneumonia requiring admission to an intensive care unit: a descriptive study.

Severe community-acquired pneumonia (CAP) requiring admission to an intensive care unit (ICU) has been inadequately studied. We compared characteristics and outcomes of patients with CAP who were admitted to the ICU with those of patients managed on the ward. Of the 3675 patients hospitalized with CAP, 374 (10%) were admitted to the ICU. The main reason for ICU admission was respiratory failure requiring intubation and ventilation (n = 303, 81%), although this indication decreased with increasing age (p < 0.05 for trend). Most patients (62%) required mechanical ventilation for 3 days or less. The following factors were predictive of ICU admission on multivariable analysis: younger age, smoker, limitation of functional status, absence of cough or pleurisy, presence of chronic obstructive pulmonary disease, substance abuse, elevated serum creatinine, abnormal serum glucose concentration, and a respiratory rate of <16 or >24 breaths per minute. Patients with low Pneumonia Severity Index scores and low CURB-65 scores were admitted to the ICU based on clinical judgment that appeared to supersede objective scoring. Severe CAP requiring admission to the ICU is common, and the decision about which patients to admit often requires clinical judgment that in many cases appears at odds with various validated pneumonia severity scoring systems.

Pneumonia during pregnancy.

BACKGROUND: We determined the incidence, clinical course, risk factors, and outcomes of community-acquired pneumonia complicating pregnancy. METHODS: A prospective study was performed of pregnant and nonpregnant patients in the same age range who presented to any of 6 hospitals in Edmonton, Alberta, with signs and symptoms of pneumonia together with an acute infiltrate evident on chest radiography compatible with pneumonia. RESULTS: There were 28 patients with pneumonia during pregnancy, for an incidence of 1.1 per 1000 deliveries, whereas there were 333 nonpregnant females in the 20- to 40-year age group with pneumonia, for an incidence of 1.3 per 1000. No significant differences in signs and symptoms were present between the two groups. Asthma requiring treatment was present in 46.4% of the pregnant patients, compared with 17.1% of the nonpregnant patients. No maternal or fetal deaths were noted except an abortion at 10 weeks of gestation. No anomaly was detected among newborns. Two patients had preterm deliveries. CONCLUSIONS: Pneumonia is well tolerated during pregnancy. Asthma may be a predisposing factor for pneumonia in this group of patients.

Differences in the features of aspiration pneumonia according to site of acquisition: community or continuing care facility.

OBJECTIVES: To determine the prevalence of aspiration pneumonia and to compare the features and risk factors for this entity in patients from continuing care facilities (CCFs) and the community who were admitted to the hospital with pneumonia. DESIGN: Prospective population-based study. SETTING: Six hospitals in Capital Health Region (Edmonton), Alberta, Canada. PARTICIPANTS: One thousand nine hundred forty-six adults admitted with pneumonia. Patients were stratified by their residence as community or CCF. MEASUREMENTS: Aspiration pneumonia prevalence; risk factors; and outcomes such as mortality, length of stay, and intensive care unit admission rates. RESULTS: Ten percent of those with community-acquired pneumonia (CAP) had aspirated, compared with 30% of those with CCF-acquired pneumonia. Those with community-acquired aspiration pneumonia (CAAP) and those with CCF-acquired aspiration pneumonia (CCF-AP) were younger, more likely to go to ICU, and more likely to require mechanical ventilation and had a longer length of stay and a higher mortality rate than nonaspirators. The risk factors for aspiration differed; for those with CAAP, impaired consciousness due to alcohol, drugs, or hepatic failure predominated, whereas 72% of those with CCF-AP had neurological disease that resulted in dysphagia. Eighty percent were treated with antibiotics effective against anaerobic bacteria. CONCLUSION: Aspiration pneumonia is common in patients with both CAP and CCF-acquired pneumonia. The risk factors differ, and there is a high mortality rate. Neurological disease dominates as the predisposing factor toward aspiration pneumonia in people in CCFs.

Tuberculous peritonitis--report of 30 cases and review of the literature.

In this retrospective study, clinical presentation of peritoneal tuberculosis in an area with a high prevalence of tuberculosis is discussed. Thirty cases diagnosed in an eight-year period are presented. The disease was 2.3 times more common in females. Eighty per cent of the patients were less than 40 years old. Excluding two cases of long duration (one year and three years), mean duration of the symptoms before admission was 2.3 months. Fever, abdominal pain, swelling and weight loss were the main symptoms. Ascites was clinically detected in 80 per cent of cases. Intermediate strength PPD tuberculin skin test was positive in 61 per cent of cases. In 57.7 per cent there was radiologic evidence of pleuropulmonary disease suggestive of tuberculosis. Fourteen of 15 ascitic fluids examined were exudative in nature with predominance of lymphocytes. In the majority of patients diagnosis was proven with laparotomy and laparoscopy (12 cases in each group). In the remainder, tissue for diagnosis was obtained through blind peritoneal needle biopsy, percutaneous biopsy of liver and peripheral lymph node biopsy (two cases with each method).