Dataset Information

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets.

ABSTRACT: There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7-11 days at 12°C in media supplemented with various additives. Effects were evaluated by calcein staining of live cells as well as morphology. Twenty-six additives were tested using (1) a two-level factorial design in which 10 additives were added or omitted in 64 different combinations and (2) a mixture design with 5 additives at 5 different concentrations in a total of 64 different mixtures. Automated microscopy and cell counting with Fiji enabled efficient processing of data. Significant regression models were identified by Design-Expert software. A calculated maximum increase of live cells to 37?±?6% was achieved upon storage of cell sheets for 11 days in the presence of 6% glycerol. The beneficial effect of glycerol was shown for epidermal cell sheets from three different donors in two different storage media and with two different factorial designs. We have thus developed a high throughput screening system enabling robust assessment of live cells and identified glycerol as a beneficial additive that has a positive effect on epidermal cell sheet upon storage at 12°C. We believe this method could be of use in other cell culture optimization strategies where a large number of conditions are compared for their effect on cell viability or other quantifiable dependent variables.

SUBMITTER: Reppe S

PROVIDER: S-EPMC6276401 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets.

Reppe Sjur S Jackson Catherine Joan CJ Ringstad Håkon H Tønseth Kim Alexander KA Bakke Hege H Eidet Jon Roger JR Utheim Tor Paaske TP

Stem cells international 20181118

There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7-11 days at 12°C in media supplemented with various additives. Effects ...[more]

PMID: 30581473

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Project description:The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

Project description:BACKGROUND:Smoking remains a leading cause of preventable death and illness. Internet interventions for smoking cessation have the potential to significantly impact public health, given their broad reach and proven effectiveness. Given the dose-response association between engagement and behavior change, identifying strategies to promote engagement is a priority across digital health interventions. Text messaging is a proven smoking cessation treatment modality and a powerful strategy to increase intervention engagement in other areas of health, but it has not been tested as an engagement strategy for a digital cessation intervention. OBJECTIVE:This study examined the impact of 4 experimental text message design factors on adult smokers' engagement with an internet smoking cessation program. METHODS:We conducted a 2×2×2×2 full factorial screening experiment wherein 864 participants were randomized to 1 of 16 experimental conditions after registering with a free internet smoking cessation program and enrolling in its automated text message program. Experimental factors were personalization (on/off), integration between the web and text message platforms (on/off), dynamic tailoring of intervention content based on user engagement (on/off), and message intensity (tapered vs abrupt drop-off). Primary outcomes were 3-month measures of engagement (ie, page views, time on site, and return visits to the website) as well as use of 6 interactive features of the internet program. All metrics were automatically tracked; there were no missing data. RESULTS:Main effects were detected for integration and dynamic tailoring. Integration significantly increased interactive feature use by participants, whereas dynamic tailoring increased the number of features used and page views. No main effects were found for message intensity or personalization alone, although several synergistic interactions with other experimental features were observed. Synergistic effects, when all experimental factors were active, resulted in the highest rates of interactive feature use and the greatest proportion of participants at high levels of engagement. Measured in terms of standardized mean differences (SMDs), effects on interactive feature use were highest for Build Support System (SMD 0.56; 95% CI 0.27 to 0.81), Choose Quit Smoking Aid (SMD 0.38; 95% CI 0.10 to 0.66), and Track Smoking Triggers (SMD 0.33; 95% CI 0.05 to 0.61). Among the engagement metrics, the largest effects were on overall feature utilization (SMD 0.33; 95% CI 0.06 to 0.59) and time on site (SMD 0.29; 95% CI 0.01 to 0.57). As no SMD >0.30 was observed for main effects on any outcome, results suggest that for some outcomes, the combined intervention was stronger than individual factors alone. CONCLUSIONS:This factorial experiment demonstrates the effectiveness of text messaging as a strategy to increase engagement with an internet smoking cessation intervention, resulting in greater overall intervention dose and greater exposure to the core components of tobacco dependence treatment that can promote abstinence. TRIAL REGISTRATION:ClinicalTrials.gov NCT02585206; https://clinicaltrials.gov/ct2/show/NCT02585206. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):RR2-10.1136/bmjopen-2015-010687.

Dataset Information

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets.

Publications

High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets.

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