nutrient-regulation-continuous-feeding-for-large-scale-expansion

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

University of Minnesota

A fusion protein consisting of the PA binding domain of anthrax lethal factor (LFn) and a codon optimized Hepatitis B virus large surface antigen (LHBsAg) expresses well in Escherichia coli. The LFn-LHBsAg fusion protein effectively elicits a cell-mediated immune (CMI) response to the hepatitis B viral antigens in mice.

Recombinant hepatitis B large surface antigen, successfully produced in Escherichia coli, stimulates T-cell response in mice.

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications.

Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy. Caring for children with ALL is an uncommon experience for parents without medical training. They urgently need professional assistance when their children are recovering at home. This paper documents the process of developing an Android application (app) "Care Assistant" for family caregivers of children with ALL. Key informant interviews and focus group studies were used before programming the app. The key informants and focus group members included: caregivers of children with ALL, cancer care physicians and nurses, and software engineers. We found several major challenges faced by caregivers: limited access to evidence-based clinic information, lack of financial and social assistance, deficient communications with doctors or nurses, lack of disease-related knowledge, and inconvenience of tracking treatments and testing results. This feedback was used to develop "Care Assistant". This app has eight modules: personal information, treatment tracking, family care, financial and social assistance, knowledge centre, self-assessment questionnaires, interactive platform, and reminders. We have also developed a web-based administration portal to manage the app. The usability and effectiveness of "Care Assistant" will be evaluated in future studies.

Developing "Care Assistant": A smartphone application to support caregivers of children with acute lymphoblastic leukaemia.

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols.

Smartphone applications are widely used for self-help interventions in adult cancer survivors. However, applications for parents of pediatric cancer patients are limited. We developed an application to assist parents of children with acute lymphoblastic leukemia. The aim of this study is to evaluate the app's usability and effectiveness in a preliminary way. A stepwise approach and mixed methods were used. The application was initially tested by healthcare providers, and their comments and suggestions were used to develop an updated version. This version was tested by parents of children with acute lymphoblastic leukemia. Comments and nonverbal expressions of parents were recorded during a 2-week pilot test. The qualitative study was followed by a quantitative study using audit log data from the administration portal to understand how parents use the application. Six healthcare providers and 15 parents participated. Parents gained a greater knowledge of leukemia, confidence in caregiving, social support, and information on how to reduce stress. Overall usability was rated as stable, useful, simple, and self-explanatory. No software failure occurred. Applications have the potential to support caregivers of pediatric cancer patients. We plan to address limitations and perform an empirical interventional study to examine its clinical effectiveness.

Zhaohui Geng

Medicine

University of Minnesota

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Zhaohui Geng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Medicine

University of Minnesota

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Medicine