Microcarrier Separation Devices for Continuous In-Line Bioprocessing of Adherent Cell Culture

Bioreactors are a space-efficient method of growing cells en masse for industrial operations by suspending the cells in an agitated vessel full of cell culture media. Adherent cell lines can be grown on microcarriers in bioreactors to reduce the space needed for petri dishes or flasks in the lab. One of the important factors in cell culture is changing the cell media to remove cell waste, secreted products, and/or replenish the nutrients available. Bioreactors face unique challenges with media changes, as cells should not be removed from a culture when media is changed. Currently, some labs let the cells settle to the bottom of the bioreactor for 30-45 min before decanting the spent media and refilling the tank with new media. This limits the cells’ access to oxygen and nutrients, and can cause the cells to die or stop producing a product of interest. This project provides bioreactor users with three single-use products that separate microcarriers from media in a continuous flow, to shorten the time adherent cells are outside of their ideal environment. The Honors extension of this project evaluates the adherent cell lines commonly used in industry, and calculates the stress induced on the microcarriers based on the fluid mechanics of the proposed designs. The designs selected for this project utilize gravitational and centrifugal settling with an inclined settler, lamella separator, and hydrocylone and were designed to operate with two peristaltic pumps to control flow rates, and separate a solution of dilute microcarriers. The Honors extension of this project also includes a robust explanation of the mechanism of settling for each design, and more clearly detail show the results can be used to customize a separator to address the needs of a lab. Flow rates were tested to determine the most effective inlet and permeate streams to maximize settling for high flow rates. Two prototype designs–the inclined settler and the lamella separator –had a separation efficiency greater than 99% for a moderate flow rate above 2.083 L/hr (50 L over 24 hours), and calculations of the maximum fluid shear forces were negligible compared to forces generated by bioreactor agitators. These designs can be scaled up to larger bioreactor cultures to allow for higher throughput and more efficient product separation, leading to longer bioreactor experiments with reduced risk of undue cell stress or death

Sterility and C2C12 Cell Growth Potential of Polycarbonate

The current data collection method of cell growth and death in a simulated space environment using a rotary cell culture system involves the use of disposable polystyrene. Polystyrene generates large amounts of waste and is limited by the sizes determined by manufacturers. A novel rotary cell culture system at USU required a material that could be custom machined for the purposes of the simulation. Polycarbonate is a durable material that is capable of being shaped, but little is known of its biocompatibility. The sterilization treatments of dry heat, autoclaving, ethanol, and UV radiation were used and compared to unsterilized polycarbonate to determine the effectiveness of each method of sterilization on cell viability of C2C12 cells. Upon 10 days of growth, the samples of polycarbonate were removed from growth media, cleaned with ethanol to kill remaining cells, and observed for structural defects and cytotoxicity. Dry heat and wet heat were found to be acceptable processes to sterilize the polycarbonate and encourage cell growth. Polycarbonate was proven non-cytotoxic by itself, as it did not cause cell death upon contact with attached C2C12 cells

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites

Developmental checkpoints eliminate B cells that synthesize defective immunoglobulin (Ig) heavy (HC) and light (LC) chains. The first checkpoint tests mHCs paired with VpreB/l5 in a pre-B cell receptor (pre-BCR) to determine whether the mHC will be able to bind conventional LCs to form membrane IgM. VpreB and l5 also create a sensing site that interacts with the mHC antigen-binding region complementarity-determining region (CDR)\u2013H3; however, whether this site contributes to Ig repertoire selection and function is unknown. We analyzed the amino acid content of CDR-H3s from HCs cloned from living and apoptotic pre-B cells and from IgG-antigen structures. Using a panel of DH gene\u2013targeted mice, we showed that progressively reducing CDR-H3 tyrosine content increasingly impaired pre-BCR checkpoint passage. Counting from cysteine at framework 3 position 96, we found that VpreB particularly selected for tyrosine at CDR-H3 position 101 and that Y101 also bound antigen in IgG-antigen structures. Therefore, in addition to its stabilization role in the pre-BCR, VpreB also acts as an early invariant antigen by selecting for particular CDR-H3 amino acids. These interactions shape the specificity of the IgG humoral response and may thus impose limitations on development of certain neutralizing antibodies

Playing the Public Lands Game- HONR 3020: Engaging Utah\u27s Public Lands

How to get involved with public land issues and learn what\u27s at stake. Join us as students present a guide that teaches how to locate, navigate, and participate in the various government and public processes for engaging in public lands debates

The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network

During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome

Silk garments plus standard care compared with standard care for treating eczema in children: A randomised, controlled, observer-blind, pragmatic trial (CLOTHES Trial)

© 2017 Thomas et al. Background: The role of clothing in the management of eczema (also called atopic dermatitis or atopic eczema) is poorly understood. This trial evaluated the effectiveness and cost-effectiveness of silk garments (in addition to standard care) for the management of eczema in children with moderate to severe disease. Methods and findings: This was a parallel-group, randomised, controlled, observer-blind trial. Children aged 1 to 15 y with moderate to severe eczema were recruited from secondary care and the community at five UK medical centres. Participants were allocated using online randomisation (1:1) to standard care or to standard care plus silk garments, stratified by age and recruiting centre. Silk garments were worn for 6 mo. Primary outcome (eczema severity) was assessed at baseline, 2, 4, and 6 mo, by nurses blinded to treatment allocation, using the Eczema Area and Severity Index (EASI), which was log-transformed for analysis (intention-to-treat analysis). A safety outcome was number of skin infections. Three hundred children were randomised (26 November 2013 to 5 May 2015): 42% girls, 79% white, mean age 5 y. Primary analysis included 282/300 (94%) children (n = 141 in each group). The garments were worn more often at night than in the day (median of 81% of nights [25th to 75th centile 57% to 96%] and 34% of days [25th to 75th centile 10% to 76%]). Geometric mean EASI scores at baseline, 2, 4, and 6 mo were, respectively, 9.2, 6.4, 5.8, and 5.4 for silk clothing and 8.4, 6.6, 6.0, and 5.4 for standard care. There was no evidence of any difference between the groups in EASI score averaged over all follow-up visits adjusted for baseline EASI score, age, and centre: adjusted ratio of geometric means 0.95, 95% CI 0.85 to 1.07, (p = 0.43). This confidence interval is equivalent to a difference of −1.5 to 0.5 in the original EASI units, which is not clinically important. Skin infections occurred in 36/142 (25%) and 39/141 (28%) of children in the silk clothing and standard care groups, respectively. Even if the small observed treatment effect was genuine, the incremental cost per quality-adjusted life year was £56,811 in the base case analysis from a National Health Service perspective, suggesting that silk garments are unlikely to be cost-effective using currently accepted thresholds. The main limitation of the study is that use of an objective primary outcome, whilst minimising detection bias, may have underestimated treatment effects. Conclusions: Silk clothing is unlikely to provide additional benefit over standard care in children with moderate to severe eczema. Trial registration: Current Controlled Trials ISRCTN77261365

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites

International audienceTransportation networks have become a subject of scientific interest from the second half of the twentieth century. Studies related to growth of the transportation networks after resuming its activity after thirty years have changed focus from the topological complexity of transportation network to their structural properties. In other words, topological complexity has changed into structural complexity. In this paper, this lost scientific direction will be reminded to the present society. Firstly, early studies on the topological complexity will be summarized and compared to each other. Secondly, new studies will be summarized and analyzed in terms of topological complexity. Finally, in conclusion, a means of measuring the topological complexity will be discussed and gaps in current studies will be highlighted

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101

Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies