Scale-up Method Research Articles

Population Size Estimation of People Who Use Illicit Drugs and Alcohol in Iran (2015-2016).

Estimating the number of people using illicit drugs and alcohol is necessary for informing health policy and programming. However, it is often challenging to reliably estimate the size of these marginalized populations through direct methods. In this study, we estimated the population size of these groups using the indirect Network Scale-Up (NSU) method in Iran from 2015 to 2016. Using a self-administered questionnaire, we asked 15 124 individuals (54% men) about the number of people they know who used different types of drugs at least once in the past 12 months. Prevalence estimates were reported per 100 000 population. The uncertainty level (UL) was calculated using the bootstrap method. The average age of the respondents was 33 years old, and 35.1% of them were unmarried. The most common drugs and their prevalence were as follows: opium (2534 [95% UL: 2467-2598]), hashish (849 [95% UL: 811-886]), stimulants (methamphetamine, ecstasy pills, cocaine, and Ritalin) (842 [95% UL: 802-879]), heroin/crack (578 [95% UL: 550-607]), and drug injection (459 [95% UL: 438-484]). Additionally, we estimated the prevalence of alcohol use as 2797 (95% UL: 2731-2861). On average, substance use was 5.23 times more prevalent among men than women. Opium use was more prevalent among individuals aged >50 years old. Moreover, alcohol use was more prevalent among participants between 18 and 30 years old (5164 per 100 000 population). Although opium continues to be the most prevalent illicit drug in Iran, the patterns of illicit drug use are heterogeneous among different age groups, genders, and provinces. Age-gender specific and culturally appropriate interventions are warranted to meet the needs of people in different subgroups.

Green, versatile, and scale-up synthesis of amides by aerobic oxidative amination over Ag2O/P-C3N4 photocatalyst

Being extensively applied in various fields of chemistry and chemical industry, the development of a green and versatile method for the synthesis of amides is in line with the demand of sustainable chemistry. Herein, a direct, highly selective, and scale-up (5–20 mmol) method for photocatalytic synthesis of amides through aerobic oxidative amination of alcohols with amines was developed under visible light, room temperature, using air as the oxidant. Benefiting from the adsorption of sodium hemiaminal on catalyst lengthens the CH bond (1.148 Å), this novel process is feasible for a broad range of functionlized amides (69 examples), especially those for drug manufacture (e.g., moclobemide and pipobroman). Imines was almost prevented with excellent amide selectivity up to 99 % could be ascribed to the low energy barrier for hemiaminal dehydrogenation while that for dehydration is high (2.13 eV). This green and efficient protocol represents an ideal alternative to the currently known methods.

Comparison of Different Methods to Estimate Canopy Water Storage Capacity of Two Shrubs in the Semi-Arid Loess Plateau of China

The canopy water storage capacity of vegetation has great significance for the hydrological cycle. We used the Pereira regression analysis method, scale-up method, and simulated rainfall method to determine canopy water storage capacity from 2014 to 2018. The Pereira regression analysis was affected mainly by the seasonal variation in the leaf area index and the observation method of throughfall. The canopy water storage capacity was 0.68 mm and 0.72 mm for C. korshinskii and H. rhamnoides, respectively. The canopy water storage capacity of C. korshinskii and H. rhamnoides was 0.73 mm and 0.76 mm, respectively, using the scale-up method. The scale-up method showed that water storage capacity per area of the canopy components was in the order of branches (0.31 mm) > leaves (0.27 mm) > trunks (0.15 mm) for C. korshinskii, and trunks (0.33 mm) > branches (0.29 mm) > leaves (0.14 mm) for H. rhamnoides. We used eight simulated rainfall intensities to determine the canopy water storage capacity for C. korshinskii and H. rhamnoides, which was 0.63 mm and 0.59 mm, respectively.

Survey Methods for Estimating the Size of Weak-Tie Personal Networks

Researchers increasingly use aggregate relational data to learn about the size and distribution of survey respondents’ weak-tie personal networks. Aggregate relational data are collected by asking questions about respondents’ connectedness to many different groups (e.g., “How many teachers do you know?”). This approach can be powerful, but to use aggregate relational data, researchers must locate external information about the size of each group from a census or administrative records (e.g., the number of teachers in the population). This need for external information makes aggregate relational data difficult or impossible to collect in many settings. Here, the authors show that relatively simple modifications can overcome this need for external data, significantly increasing the flexibility of the method and weakening key assumptions required by the associated estimators. The key idea is to estimate the size of these groups from the sample of survey respondents, rather than relying on external sources of information. These methods are appropriate for using a sample survey to study the size and distribution of weak-tie network connections. They can also be used as part of the network scale-up method to estimate the size of hidden populations. The authors illustrate this approach with two empirical studies: a large simulation study and original household survey data collected in Hanoi, Vietnam.

A Review of Prevalence Estimation Methods for Human Trafficking Populations.

Human trafficking has long-lasting implications for the well-being of trafficked people, families, and affected communities. Prevention and intervention efforts, however, have been stymied by a lack of information on the scale and scope of the problem. Because trafficked people are mostly hidden from view, traditional methods of establishing prevalence can be prohibitively expensive in the recruitment, participation, and retention of survey participants. Also, trafficked people are not randomly distributed in the general population. Researchers have therefore begun to apply methods previously used in public health research and other fields on hard-to-reach populations to measure the prevalence of human trafficking. In this topical review, we examine how these prevalence methods used for hard-to-reach populations can be used to measure the prevalence of human trafficking. These methods include network-based approaches, such as respondent-driven sampling and the network scale-up method, and venue-based methods. Respondent-driven sampling is useful, for example, when little information about the trafficked population has been produced and when an adequate sampling frame does not exist. The network scale-up method is unique in that it does not target the hidden population directly. The implications of our work internationally include the need for documenting and validating the various prevalence estimation methods in the United States in a more robust way than was done in existing efforts. In providing this roadmap for estimating the prevalence of human trafficking, our overarching goal is to promote the equitable treatment and overall well-being of the socially disadvantaged populations who disproportionately experience human trafficking.

Development and optimization of the novel scale-up fabrication method of polyvinylpyrrolidone (PVP) microparticles

This research proposed a new method for making polymer microparticles. Polyvinylpyrrolidone was used in concentrations of 1–5%. First, the critical conditions of microparticles were evaluated through the electrocentrifuge spinning system. The critical voltage, rotating speed, and solution concentration were obtained. The results showed that the number of particles increased in each solution concentration and the particle size decreased with increasing the voltage. Also, as the rotating speed increases, the particles reach the bed before drying, thus sticking to each other and creating a smooth surface. However, at high voltages, the particles stick less to each other.

Estimation of Injuries in a Population-based Study by Stratified Cluster Sampling and Network Scale-up Methods

Introduction: Today, one of the most important causes of morbidity and mortality among people all across the world is Injury. Injuries pose an important challenge for the health system and account for 28% of the deaths per day in Iran. Iran is in the 5th place for road traffic death rates globally.
 Objective: This was a cross sectional study aimed to estimate annual incidence rates of injury in individuals above 15 in Kashan, Iran.
 Methods: In this population-based cross-sectional study, people above 15 years in households residing in Kashan during 2018-2019 were studied The sample had a twofold design, Stratified Cluster Sampling method and network Scale-up method. Data analysis was performed using SPSS 22 (Chi-square and T-test and Logistic regression analysis) and Stata 14 software.
 Results: This study describes the study setting, sampling, data collection and management, quality control and assurance, and the statistical analysis. In this study, the annual incidence rate from all kinds of trauma was estimated at 70.61(62.60-78.70) per 1000 person-years, and the death caused by injury was 4.63(34.32-56.12) per 1000 person-years. In addition, in network scale-up method incidence rate of all kinds of trauma was 57.1 and the death caused by trauma was 3.47 per 1000 person-years.
 Conclusion: population-based studies were used to evaluate injuries and outcomes of trauma, so that managers realize where attention is needed regarding preventive programs in this city. Using indirect sampling techniques can be cost-effective and time-consuming, and can provide us with the information urgently needed.

One-step synthesis of nanoscale zero-valent iron modified hydrophobic mesoporous activated carbon for efficient removal of bulky organic pollutants

It is of vital importance for industrial applications by developing simple, robust, economic, eco-friendly and easy scale-up methods for fabricating hydrophobic mesoporous activated carbon (MAC) from sustainable raw materials with high adsorption performance for bulky organic pollutants removal. Herein, a novel one-step facile approach coupling carbothermal reduction with catalytic activation was firstly established for preparing the coconut shell-based hydrophobic MAC modified with nanoscale zero-valent iron (NZVI@MAC). The in-situ synthesized NZVI (merely 0.33 wt%) by carbothermal reduction could not only catalyze the gasification reaction between carbon and CO2 to fabricate MAC (dBET = 5.17 nm, SBET = 933.12 m2 g−1), but also catalyze the amorphous carbon to form hydrophobic graphitic layers covering its surface. In striking contrast to the unmodified activated carbon, the NZVI@MAC surprisingly exhibited a remarkably 8.62-fold higher initial adsorption rate and a slightly increased adsorption capacity of 327.60 mg g−1 for bisphenol A (BPA). Intriguingly, the high mesoporous proportion (70–90%) of NZVI@MAC with the average pore size of 4.1–5.2 nm was effective in reducing the diffusion resistance for BPA. Such high adsorption performance under neutral conditions was primarily attributed to the strong hydrophobic interaction, π-π interaction and the weak hydrogen bonding. Moreover, the NZVI@MAC showed robust anti-interference properties in actual environmental conditions containing various inorganic ions and in a wide pH range (3–10). Therefore, the cost-effective, eco-friendly, sustainable and recyclable hydrophobic NZVI@MAC with stable structure and excellent performance for bulky organic adsorption is a promising material for practical applications, especially in drinking water purification.

Carbon dioxide capture in gallate-based metal-organic frameworks

Carbon capture and sequestration are vital industrial processes that not only mitigate the greenhouse effect but also promote upgrading of low-quality natural gas. Porous adsorbent-based adsorption separation process appeals to be promising, while it still remains a great challenge to develop adsorbents with high adsorption uptake and selectivity of CO2 as well as high working capacity in practical applications. In this work, a group of gallate-based metal-organic frameworks with aperture size of 3.52 to 3.65 Å were reported for molecular sieving of CO2 from N2 and CH4. Remarkably, Mg-gallate exhibited a record-high uptake of CO2 (5.05 mmol g−1) among all reported molecular sieving adsorbents combined with ultrahigh uptake ratio of CO2/CH4 (29.7) and CO2/N2 (84.2) at 1 bar and 298 K. Multiple O-H…C and O…H-O interactions afford dense packing of CO2 molecules in the framework structure with a density up to 1.06 g cm−3 on Mg-gallate, verified by density function theory calculations. Experimental breakthrough experiments confirmed the superb separation performance of flue gas and natural gas over a wide range of temperature. Robust structure and cycling stability along with facile scale-up synthesis method further offer great potential of M−gallate in industrial applications.

Measure of Personal Network Size Using the Known Population Method: A Methodological Guide.

The network scale-up method (NSUM) has shown promise in measuring the prevalence of hidden public health problems and at-risk populations. The technique involves asking survey respondents how many people they know with the health problem or characteristic of interest and extrapolating this information to the population level. An important component of the NSUM estimate is the size of each respondent's network, which is determined by asking respondents about the number of people they know who belong to populations of known size. There is little systematic discussion, however, to guide selection of these questions. Furthermore, many of the most commonly used known population questions are appropriate only in countries with a robust data infrastructure. Here, we draw from the NSUM literature to present a set of best practices in the selection of NSUM known population questions. Throughout, we address the unique situations that many researchers face in collecting prevalence data in the developing world, where innovative prevalence estimation techniques, such as NSUM, are most needed. (Am J Public Health. 2022;112(5):747-753. https://doi.org/10.2105/AJPH.2022.306731).

Method to maximize the productivity of laser powder bed fusion systems through dimensionless parameters

Productivity in laser powder bed fusion systems can be increased by using high layer thickness (>40 μm). The process parameters for high layer thickness are typically found by one-factor-at-a-time, design of experiments, or computationally intensive numerical simulations. In this paper, a method to scale-up the process parameters from low to high layer thickness is proposed. The method is based on dimensionless parameters from the analytical model. Through the proposed scale-up method, the build rate increase was proportional to an increase in layer thickness. The scale-up method was demonstrated for laser powder bed fusion of stainless steel 316L from 30 to 50 μm layer thickness and from 50 to 70 μm layer thickness. For both cases, no detriment to part density was observed—measured densities before and after the scale-up were above 99.6%. The density results obtained were within high density windows with variation of parameters resulting in the same respective volumetric energy densities and one-factor-at-a-time parameter studies. There was no significant change in tensile properties after the scale-up except reduction in elongation at break in the XY direction. The comparable mechanical properties before and after the scale-up method were attributed to the observed similarities in microstructure features such as the crystal orientation, cell sizes, and proportions of low and high angle grain boundaries.

Study on the Umbilical Cord-Mesenchymal Stem Cell Manufacturing Using Clinical-Grade Culture Medium.

Mesenchymal stem/stromal cell (MSC)-based therapies have been gaining increasing attention owing to their application in various diseases and conditions. In this study, we aimed to identify the optimal condition for industrial-scale MSC manufacturing. MSCs were isolated from umbilical cord (UC) tissues by implementing the explant method (Exp) or a collagenase based-enzymatic digestion method (Col), using a good manufacturing practice-compatible serum-free medium developed in-house. Microarray analysis demonstrated that the gene expression profiles of Exp-MSCs and Col-MSCs did not significantly differ according to the method of isolation or the culture conditions used. The isolated UC-MSCs were then subjected to expansion using conventional static culture (ST) or microcarrier-based culture in stirred-tank bioreactors (MC). Metabolomic and cytokine array analyses were conducted to evaluate the biochemical status of the MSCs. However, no remarkable differences in the metabolic profile and cytokine secretome between ST-MSCs and MC-MSCs were observed. On the contrary, we observed for the first time that the hydrophobic components of ST-MSCs and MC-MSCs were different, which suggested that the cell membrane distribution of fatty acids and lipids was altered in the process of adaptation to shear stress in MC-MSCs. These results establish the flexibility of the isolation and expansion method for UC-MSCs during the manufacturing processes and provide new insights into the minor differences between expansion methods that may exert remarkable effects on MSCs. In conclusion, we demonstrated the feasibility of both Exp-MSCs and Col-MSCs and MC and ST culture methods for scale-up and scale-out of MSC production, as well as the equivalence of these cells. As for the industrialized mass production of MSCs, enzyme-based methods for isolation and cell expansion in a bioreactor were considered to be more suitable. The methods developed, which underwent comprehensive evaluation in this study, may contribute toward the provision of sufficient MSC sources and the establishment of cost-effective MSC therapies. Impact statement Our in-house-developed good manufacturing practice-grade serum-free medium could be used for both isolation (Exp and Col) and expansion (ST and MC) of umbilical cord (UC)-mesenchymal stem/stromal cells (MSCs). Characteristics of the obtained UC-MSCs were widely assessed with regard to gene expression, metabolome, and secretome. Cellular characteristics and efficacy were observed to be equivalently maintained among whichever technique was applied. In addition, our research presents the first evidence that bioreactor and microcarrier-based MSC cultures alter the fatty acid and phospholipid composition of MSCs. These results provide new insights into the differences between expansion methods that may exert remarkable effects on MSCs.

Estimating the prevalence of high-risk behaviors using network scale-up method in medical university students.

There is not much information about high-risk behaviors in young groups, especially students. This study was conducted to estimate the prevalence of high-risk behaviors in students of universities of medical sciences in West Azerbaijan Province, Iran, by network scale-up (NSU) method. This cross-sectional study was performed on 450 students from the universities of medical sciences. A researcher-developed checklist was used to collect the data. We considered number 16 for the social network size of students according to a previous study. Based on the response of individuals to each of the high-risk behaviors (including cigarette smoking, hookah use, opium consumption, alcohol drinking, tramadol/ecstasy taking, and extramarital sex) in their social network, the prevalence of these behaviors was estimated. The required calculations were performed using the NSU method. Furthermore, 95% uncertainty interval (UI) was calculated using the bootstrap method. Totally, 196 (44%) participants were male. The mean age (standard deviation) of the participants was 22 ± 2 years. Results showed that hookah use (20% 95% UI [18.9-21.1]) and opium consumption (0.4% 95% UI [0.24-0.6]) had the highest and lowest frequencies, respectively. Cigarette smoking (17% 95% UI [15.8-18]), alcohol use (8.3% 95% UI [7.5-9.1]), extramarital sex (8.2% 95% UI [7.4-9]), and tramadol/ecstasy taking (4% 95% UI [6.4-4.6]) were the next most common high-risk behaviors, respectively. Given that hookah use and cigarette smoking are the most common high-risk behaviors in students, especially males, appropriate cultural activities and educational programs should be employed by relevant authorities to reduce these behaviors.

Pilot-scale extraction of polyphenols from spent black tea by semi-continuous subcritical solvent extraction

Spent black tea (SBT) is a residue from tea beverage production and considered as a potential source of active polyphenols. This study aimed to develop a pilot-scale process on semi-continuous subcritical solvent extraction (SSE) of polyphenols from SBT by exploiting the lab-scale knowledge. Treatment of SBT with ethanol–water (50% w/w) as solvent at 125 °C and 0.3 MPa achieved a significantly higher yield of polyphenols (80.82 g gallic acid equivalents/kg black tea) with antioxidant activity (64.20 g gallic acid equivalents/kg black tea), compared to hot water extraction (HWE). SSE increased the soluble matter content in extracts than HWE. Based on the results of LC-MS, theaflavin-3,3′-digallate was the most abundant polyphenol from a total of 12 compounds to be extracted by SBT with 50% ethanol. The results suggested that SSE can be used as a scale-up extraction method to recover polyphenols from SBT.

A systematic review of adaptations and effectiveness of scaled-up nutrition interventions

ContextPublic health nutrition interventions shown to be effective under optimal research conditions need to be scaled up and implemented in real-world settings.ObjectivesThe primary aim for this review was to assess the effectiveness of scaled-up public health nutrition interventions with proven efficacy, as examined in a randomized controlled trial. Secondary objectives were to: 1) determine if the effect size of scaled-up interventions were comparable to the prescale effect, and; 2) identify any adaptations made during the scale-up process.Data sourcesSix electronic databases were searched and field experts contacted.Study selectionAn intervention was considered scaled up if it was delivered on a larger scale than a preceding randomized controlled trial (“prescale”) in which a significant intervention effect (P ≤ 0.05) was reported on a measure of nutrition.Data extractionTwo reviewers independently performed screening and data extraction. Effect size differences between prescale and scaled-up interventions were quantified. Adaptations to scale-up studies were coded according to the Adaptome model.ResultsTen scaled-up nutrition interventions were identified. The effect size difference between prescale trials and scaled-up studies ranged from –32.2% to 222% (median, 50%). All studies made adaptations between prescale to scaled-up interventions.ConclusionThe effects of nutrition interventions implemented at scale typically were half that achieved in prior efficacy trials. Identifying effective scale-up strategies and methods to support retainment of the original prescale effect size is urgently needed to inform public health policy.Systematic Review RegistrationPROSPERO registration no.CRD42020149267.

Practical implementation of a method for global single-phase flow-based transmissibility upscaling using generic flow boundary conditions and its application on models with non-local heterogeneities

Local upscaling techniques fall short in capturing the dynamic effects of laterally continuous non-local geologic heterogeneities, for example, high-permeability channels, vertically thin, yet, horizontally extensive shale layers. Global upscaling techniques have been introduced to remedy the shortcomings of local scale-up approaches. In order to address the upscaling challenges associated with non-local heterogeneities, we have developed a practical and effective flow-based transmissibility scale-up method that computes coarse-scale transmissibilities using flux derived from single-phase flow simulations with generic global boundary conditions, referred to as the global transmissibility upscaling (GTU) method. GTU targets more accurate upscaling of laterally continuous non-local heterogeneities including the ones modeled on distorted cornerpoint grids. A variant of GTU is developed in this context that uses fluxes computed by use of the multi-point flux approximation (MPFA) discretization technique at the fine scale. While various global upscaling methods were described in the literature, they are rarely used in industrial applications due to challenges related to implementation. We describe in detail an effective practical implementation of the GTU method in a comprehensive industrial-grade simulation package and share our learnings with GTU on aggressively upscaled models with non-local heterogeneities. We demonstrate the application of GTU to several challenging reservoir models. Comparisons of GTU-based model responses are made with ones stemming from local upscaling techniques popularly utilized in the industry using fine-scale model predictions as reference. Investigated numerical examples demonstrate that the GTU method leads to notable improvements in the accuracy delivered by coarse-scale models. Especially, the application of GTU is most impactful for high-upscaling-ratio models with complex fine-scale connectivity because GTU preserves the fine-scale behavior more authentically compared to local techniques. We demonstrate that GTU naturally complements an innovative regional multiphase upscaling (RMU) method on aggressively upscaled models with strong multiphase flow associated with displacement-type recovery mechanisms. It has also been observed that using MPFA on cornerpoint grids to obtain fine-scale fluxes for GTU gives rise to more accurate coarse-scale pressure behavior. We have also observed that GTU is especially effective when combined with a regional multiphase upscaling method for ultimate accuracy in simulations of displacement-based recovery mechanisms. • A practical global transmissibility upscaling method using global flows enables fast and accurate flow simulations. • Global transmissibility upscaling preserves the fine-scale information more authentically compared to local techniques. • Global transmissibility upscaling combined with regional multiphase upscaling for fast and accurate flow simulation. • Speed-up values in the order of 10–50 fold are achieved over fine-scale model with a good compromise of accuracy.

An improved extraction and purification method for obtaining high-quality chitin and chitosan from blue swimmer (Portunus pelagicus) crab shell waste.

The online version contains supplementary material available at 10.1007/s10068-021-01002-x.

Extruded Mesenchymal Stem Cell Nanovesicles Are Equally Potent to Natural Extracellular Vesicles in Cardiac Repair.

Mesenchymal stem cells (MSCs) repair injured tissues mainly through their paracrine actions. One of the important paracrine components of MSC secretomes is the extracellular vesicle (EV). The therapeutic potential of MSC-EVs has been established in various cardiac injury preclinical models. However, the large-scale production of EVs remains a challenge. We sought to develop a scale-up friendly method to generate a large number of therapeutic nanovesicles from MSCs by extrusion. Those extruded nanovesicles (NVs) are miniature versions of MSCs in terms of surface marker expression. The yield of NVs is 20-fold more than that of EVs. In vitro, cell-based assays demonstrated the myocardial protective effects and therapeutic potential of NVs. Intramyocardial delivery of NVs in the injured heart after ischemia-reperfusion led to a reduction in scar sizes and preservation of cardiac functions. Such therapeutic benefits are similar to those injected with natural EVs from the same MSC parental cells. In addition, NV therapy promoted angiogenesis and proliferation of cardiomyocytes in the post-injury heart. In summary, extrusion is a highly efficient method to generate a large quantity of therapeutic NVs that can potentially replace extracellular vesicles in regenerative medicine applications.

Generating Large Numbers of Human Megakaryocytes from Pluripotent Stem Cells

The process of hemostasis and blood coagulation relies heavily on a sufficient supply of platelets (PLTs, also known as thrombocytes) within a person's bloodstream. Platelet transfusion is an effective treatment for thrombocytopenia-related diseases, yet paucity of supply and limited shelf-life (5 - 7 days) remain challenging. PLTs are generated by the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) into megakaryocytes (MKs), a rare subset of large polyploid bone marrow cells. Methods to produce MKs in vitro by inducing mesodermal specification and hematopoietic differentiation of human pluripotent stem cells (hPSCs) could provide a reliable and safe supply of PLTs for transfusion and would also be amenable to gene editing for generation of HLA-null universal PLTs. Culture methods to generate hPSC-derived MKs and PLTs have been described, yet published protocols lack standardization, are PSC line dependent and/or promote differentiation of other lineages, resulting in low MK cell yields and purity. The use of feeder cells and/or viral vectors also limits the clinical and scale-up applicability. Here, we describe an efficient feeder cell-free and serum-free culture system that promotes the selective differentiation of hPSCs from multiple ES and iPS lines into polyploid MKs with high purity and yields and ability to generate platelets.The 17-day protocol includes two stages: a 12-day stage to differentiate hPSCs into megakaryocytic-biased HSPCs through endothelial-to-hematopoietic transition (H-phase), and a 5-day stage to further differentiate HSPCs into mature MKs (MK-phase). at the start of the H-phase, hPSC aggregates were plated in mTeSR TM media on Matrigel ®-coated plates at 16 aggregates (100 - 200 µm in diameter, ~100 cells per aggregate) per cm 2 to allow attachment overnight (Day -1). The cells were then cultured in mesoderm-induction medium for 3 days (Day 0 - 3), and subsequently in hematopoietic specification medium for 9 days (Day 3 - 12). During this phase, PSC-derived HSPCs emerged from an adherent layer of endothelial cells and were released into suspension. On day 12 these nonadherent cells were harvested and seeded at 1 - 3.5 × 10 5 cells/mL in MK maturation medium containing thrombopoietin (TPO) and other hematopoietic growth factors and cultured for 5 additional days (MK-phase, Day 12 - 17). At the end of H-phase (day 12) and MK-phase (day 17) the cells were counted and assessed for HSPC markers (CD34/CD45), the erythroid marker glycophorin A (GlyA), MK markers (CD41a/CD42b), DNA ploidy profile and PLT production by flow cytometry and immunofluorescence microscopy. Two embryonic stem (ES) cell lines (H1 and H9), and two induced pluripotent stem (iPS) cell lines (WLS-1C and STiPS-R038) were used in this study.At the end of H-phase (Day 12), on average 48% (range: 34 - 72%) of cells released into suspension co-expressed CD41a and CD42b markers, with an average yield of 93 CD41a +CD42b + cells per seeded hPSC (range: 30 - 200, n = 4 for H9/1C, n = 3 for H1/R038). The cells also expressed CD34 (average of 78% CD34 + cells) and GlyA (average of 71% GlyA + cells), indicating that the H-phase may support differentiation of PSCs to megakaryocyte-erythroid progenitors. At the end of MK-phase (Day 17), on average 82% of the cells expressed CD41a (range: 70 - 99%), 62% of the cells co-expressed CD41a and CD42b (range: 40 - 85%), and an average of 253 CD41a +CD42b + cells were generated per seeded hPSC (range: 70 - 700 MKs, n = 11 for H1/H9/R038, n = 7 for 1C). Of note, less than 5% of cells expressed GlyA, showing that the culture system is specific for megakaryocytic differentiation. The DNA ploidy profile of the CD41a +CD42b + cells generated on Day 17 showed that on average 26% and 9% of cells had 4N and 8N+ DNA ploidy, respectively (n = 11). Multinucleated MKs could also be readily observed by immunofluorescence microscopy. These PSC-derived MKs produced an average of 3.5 PLTs (range: 1 - 10 PLTs, n = 11) based on viable CD41a +CD45 -GlyA - PLT-like particles with a similar size and CD41 expression as control PLTs prepared from fresh blood.In conclusion, we have developed a simple two-step, yet robust serum- and feeder-free culture system for generating high numbers of hPSC-MKs that are large, polyploid, and capable of shedding PLTs. This culture method provides a platform to study thrombopoiesis and is amenable to scale-up method development. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of highly water‐absorbing chromium oxyhydroxide as a dopant in a hybrid proton‐conducting membrane

Proton-conductive membranes restrict the energy conversion efficiency and operating performance of the proton exchange membrane fuel cells. In this work, we develop a simple and easy scale-up method to synthesise a new proton-conductive material, amorphous chromium oxyhydroxide (CrO(OH)·xH2O). The CrO(OH)·xH2O shows a high water-adsorption value of 450 mg g−1, contributing to the construction of continuous and ordered hydrogen-bonding network within the materials. An ultra-high proton conductivity of 0.31 S cm−1 is obtained in CrO(OH)·xH2O at 90°C and 98% relative humidity, with an activation energy of 0.39 eV, which indicates a proton hopping mechanism. To further study the application potential of CrO(OH)·xH2O in proton exchange membranes, CrO(OH)·xH2O is added to sulfonated poly(ether ether ketone) (SPEEK) as a dopant, forming a SPEEK-CrO(OH) proton exchange membrane. The composite membrane exhibits a proton conductivity comparable to Nafion, 0.17 S cm−1 at 80°C in water without decreasing in 36 hours. This research enlightens us the development of metal oxyhydroxide via a simple and feasible strategy as efficient proton conductors.