Quartz crystal microbalance as an assay to detect anti-drug antibodies for the immunogenicity assessment of therapeutic biologics.

Because of their biological origins, therapeutic biologics can trigger an unwanted deleterious immune response with some patients. The immunogenicity of therapeutic biologics can affect drug efficacy and patient safety by the production of circulating anti-drug antibodies (ADA). In this study, quartz crystal microbalance (QCM) was developed as an assay to detect ADA. Etanercept (Enbrel®) was covalently grafted to dextran-modified QCM surfaces. Rabbits were immunized with etanercept to generate ADA. Results showed the QCM assay could detect purified ADA from rabbits at concentrations as low as 50 ng/mL, within the sensitivity range of ELISA. The QCM assay could also assess the ADA isotype. It was shown that the ADA were composed of the IgG isotype, but not IgM, as expected. Furthermore, it was shown that QCM surfaces that had been used to detect ADA could be regenerated in glycine-HCl solution and reused. The QCM assay was also demonstrated to detect ADA in crude serum samples. Serum was collected from the rabbits and analyzed before and after etanercept immunization. ADA were clearly detected in serum from rabbits after immunization, but not in serum before immunization. Serum from patients administered with etanercept for rheumatoid arthritis (RA) treatment was also analyzed and compared to serum from healthy donors. Sera from 10 RA patients were analyzed. Results showed one of the RA patient serum samples may have ADA present. In conclusion, QCM appears to be a viable assay to detect ADA for the immunogenicity assessment of therapeutic biologics.

Intracellular insulin quantification by cell-ELISA.

Current methods of monitoring insulin in culture are limited to soluble insulin (secretions or lysates) or synthetic gene reporter analyses. We present an insulin-specific cell enzyme-linked immunosorbent assay (cell-ELISA) to assess relative intracellular insulin protein content of adherent cultures, normalized to cell density with further immunocytochemical verification of insulin-expressing cells within identical cultures. The protocol was optimized and validated using an insulin-expressing cell line (INS-1) by confirming direct relations between intracellular insulin content and insulin-expressing cell density, in a glucose exposure-dependent manner. Utility was demonstrated by identifying multiple INS-1 clones lowly expressing insulin following lentiviral particle delivery of interference RNA intended to down-regulate one of the insulin gene-directed transcription factors, MafA. The cell-ELISA was also applied to monitor insulin content within partially dissociated primary mouse islet cultures. This technique allows for the first time routine analysis of intracellular insulin protein in vitro suitable for investigating islet cell biology by means of multiple parameter screening.

A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

Decellularized bladder as scaffold to support proliferation and functionality of insulin-secreting pancreatic cells.

Loss in the number or function of insulin-producing ß-cells in pancreatic islets has been associated with diabetes mellitus. Although islet transplantation can be an alternative treatment, complications such as apoptosis, ischaemia and loss of viability have been reported. The use of decellularized organs as scaffolds in tissue engineering is of interest owing to the unique ultrastructure and composition of the extracellular matrix (ECM) believed to act on tissue regeneration. In this study, a cell culture system has been designed to study the effect of decellularized porcine bladder pieces on INS-1 cells, a cell line secreting insulin in response to glucose stimulation. Porcine bladders were decellularized using two techniques: a detergent-containing and a detergent-free methods. The resulting ECMs were characterized for the removal of both cells and dsDNA. INS-1 cells were not viable on ECM produced using detergent (i.e., sodium dodecyl sulfate). INS-1 cells were visualized following 7 days of culture on detergent-free decellularized bladders using a cell viability and metabolism assay (MTT) and cell proliferation quantified (CyQUANT™ NF Cell Proliferation Assay). Further, glucose-stimulated insulin secretion and immunostaining confirmed that cells were functional in response to glucose stimulation, as well as they expressed insulin and interacted with the detergent-free produced ECM, respectively.

A model for cellulase production from Trichoderma reesei in an airlift reactor.

A mathematical model for cellulase production by Trichoderma reesei RUT-C30 grown in a cellulose medium with lactose as fed batch in an airlift reactor is proposed. To describe adequately the mass transfer between the air bubbles and the broth, it uses computational fluid dynamics (CFD) including multiphase Eulerian-Eulerian formulation, with a detailed description of the bubble size distribution through the use of the population balance model (PBM) and the class method (CM). The kinetics of the biomass growth is further coupled to the fluid flow conditions using partial differential equations for all the species involved, providing detailed information of important reactor conditions such as the distribution of oxygen, cellulose, and the shear stress within the reactor over the entire period of fermentation. Predicted results agree well with the available overall measurements for a typical fed-batch operation and detailed profiles of the predicted concentration fields are discussed from an engineering viewpoint.

Bioreactor controlled by PI algorithm and operated with a perfusion chamber to support endothelial cell survival and proliferation.

This paper reports the optimization of a perfusion bioreactor system previously reported by us (Chouinard et al., 2009). The implementation of a proportional-integral (PI) controller algorithm to control oxygen concentration and pH is presented and discussed. P and I values used by the controller were first estimated using a First-Order-Plus-Dead-Time (FOPDT, Matlab Simulink) and then tuned manually. A new gas exchanger design compatible with the PI controller was introduced and validated to decrease interaction between the injected gases and overall inertia of the system. The gas exchanger was used to adjust both pH and dissolved oxygen (DO) concentration. This new bioreactor system allowed real-time PI control over pH and DO concentration at different flow rates (from 2 to 70 mL min(-1)). Cell viability and proliferation were investigated to validate the updated bioreactor design and performance.

Endothelial cell responses towards low-fouling surfaces bearing RGD in a three-dimensional environment.

This study reveals that it is possible to obtain a specific cell response towards low-fouling carboxymethyl dextran (CMD) surfaces bearing the RGD adhesive peptide in fibrin. To avoid cell sedimentation on surfaces observed in traditional cell culture systems, CMD surfaces bearing RGD were vertically embedded in fibrin containing human umbilical vein endothelial cells (HUVEC) and their effect over cells was investigated. Compared to the CMD surfaces and to CMD layers bearing the negative control RGE, RGD coatings promoted cell adhesion, induced focal contact formation indicated by co-localization of vinculin and actin fibers, and presented a significant effect over HUVEC net growth during the first 24h of the culture, as revealed by Ki67 staining and cell counting. The intracellular localization of caveolin-1 combined with the expression of beta 1 integrins was investigated and the orientation of HUVEC towards and on the RGD surfaces was studied. When compared to the negative controls, HUVEC responded to the RGD surface in fibrin resulting in acceleration of morphological changes. RGD surfaces supported fibrin degradation by HUVEC as revealed by fluorescent fibrin experiments as well as multi-cellular structure formation, vacuolation and lumen formation.

Overview of approval procedures for bioadhesives in the United States of America and Canada.

Bioadhesives are useful medical devices to help reduce postoperative complications and as adjuncts to sutures and staples in sealing wounds. Biomedical companies have been promoting research and development into new bioadhesives. As for other medical devices, translating promising candidates to market involves the need to pass through several regulatory steps, wherein their safety and effectiveness are evaluated and the proper reimbursements from payors are assessed. The regulatory procedures involve classification based on the risk factors, support studies, submission of applications to relevant authorities, procurement of certification, and finally commercialization, while keeping a track record of the post-market data. The importance of real-world data has been recently realized. The aim of this review is to focus on the translational goals, expectations, and necessities of medical devices focusing on the bioadhesives to be commercialized. It should aid researchers inspired to discover and market new bioadhesives in understanding the need for basic regulatory procedures behind their commercialization for medical usage, most importantly for internal medicine specifically in the United States of America, Canada, and Europe, in part. The key differences in the regulatory aspects among those are highlighted. Regulations keep changing with the introduction of new products and governmental laws. They are updated in this manuscript till March 2021.

Composition, host responses and clinical applications of bioadhesives.

Bioadhesives are medical devices used to join or seal tissues that have been injured or incised. They have been classified into tissue adhesives, sealants, and hemostatic agents. Bioadhesives such as FloSeal®, CoSeal®, BioGlue®, Evicel®, Tisseel®, Progel™ PALS, and TissuGlu® have been commercialized and used in clinical setting. They can be formulated with natural or synthetic components or a combination of both including albumin, glutaraldehyde, chitosan, cyanoacrylate, fibrin and thrombin, gelatin, polyethylene glycol (PEG), along with urethanes. Each formulation has intrinsic properties and has been developed and validated for a specific application. This review article briefs the mechanisms by which bioadhesives forms adhesion to tissues and highlights the correlation between bioadhesives composition and their potential host responses. Furthermore, clinical applications of bioadhesives and their application-driven requirements are outlined.

Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture chamber.

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close-loop tubular reactor. A dispersion model in one dimension has been used to describe the non-ideal behavior of the reactor. From open-loop experiments (single-cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min(-1)) had no effect over RTD function expressed in reduced time (θ). Lower flow rates (5 and 15 mL min(-1)) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non-significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Per . Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL(-1) had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments.

Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains.

BACKGROUND: Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. RESULTS: Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. CONCLUSIONS: This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.

Characterization of three-dimensional rat central nervous system culture maturation, with applications to monitor cholinergic integrity.

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H2 O2 , glutamate, or amyloid ß(1-42); each resulted in diminished AChE activity. H2 O2 exposure resulted in nuclear fragmentation. Glutamate and amyloid ß(1-42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures.

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real-time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48-h culture period. Cells were uniformly dispersed within the 14.40 mm x 17.46 mm x 6.35 mm chamber. A larger fraction of the cells suspended in 6.35-mm thick gels and cultured in a traditional CO(2) incubator were found to be round and dead [corrected]. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel.

Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells.

There is increasing evidence suggesting that oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury contributing to the age-related physio-pathological process of atherosclerosis. In this study, the effects of native LDL and ox-LDL on the mechanical properties of living human umbilical vein endothelial cells (HUVEC) were investigated by atomic force microscopy (AFM) force measurements. The contribution of filamentous actin (F-actin) and vimentin on cytoskeletal network organization were also examined by fluorescence microscopy. Our results revealed that ox-LDL had an impact on the HUVEC shape by interfering with F-actin and vimentin while native LDL showed no effect. AFM colloidal force measurements on living individual HUVEC were successfully used to measure stiffness of cells exposed to native and ox-LDL. AFM results demonstrated that the cell body became significantly stiffer when cells were exposed for 24 h to ox-LDL while cells exposed for 24 h to native LDL displayed similar rigidity to that of the control cells. Young's moduli of LDL-exposed HUVEC were calculated using two models. This study thus provides quantitative evidence on biomechanical mechanisms related to endothelial cell dysfunction and may give new insight on strategies aiming to protect endothelial function in atherosclerosis.

Method for isolation of pancreatic blood vessels, their culture and coculture with islets of langerhans.

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 µm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.

The role of elastin-derived peptides in human physiology and diseases.

Once considered as inert, the extracellular matrix recently revealed to be biologically active. Elastin is one of the most important components of the extracellular matrix. Many vital organs including arteries, lungs and skin contain high amounts of elastin to assure their correct function. Physiologically, the organism contains a determined quantity of elastin from the early development which may remain physiologically constant due to its very long half-life and very low turnover. Taking into consideration the continuously ongoing challenges during life, there is a physiological degradation of elastin into elastin-derived peptides which is accentuated in several disease states such as obstructive pulmonary diseases, atherosclerosis and aortic aneurysm. These elastin-derived peptides have been shown to have various biological effects mediated through their interaction with their cognate receptor called elastin receptor complex eliciting several signal transduction pathways. In this review, we will describe the production and the biological effects of elastin-derived peptides in physiology and pathology.

Physico-chemical properties and cytotoxicity assessment of PEG-modified liposomes containing human hemoglobin.

PEGylated liposomes encapsulating human hemoglobin as oxygen carriers were prepared from purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DMPE-PEG(2000)) and palmitic acid. Hemoglobin was extracted and purified from human blood samples. SDS-PAGE was used to assess its purity. Diameter of liposomes containing hemoglobin was controlled to approximately 200 nm using extrusion as measured by dynamic light scattering and transmission electron microscopy. Liposome size distributions were shown to remain unimodal over 14 days, even at different storage temperatures. Zeta potential measurements revealed that liposome containing hemoglobin have a net surface charge of -7.16+/-0.33 mV. Also, hemoglobin encapsulated in liposomes was able to perform several cycles of oxygen loading and unloading using oxygen (O(2)) and carbon monoxide (CO). The hemoglobin vesicle dispersion showed some toxicity as revealed by three in vitro assays in which endothelial cell (HUVECs) monolayers were exposed to these dispersions. Cytotoxicity was function of the liposome concentration in the culture medium.

Decellularized pancreas as a native extracellular matrix scaffold for pancreatic islet seeding and culture.

Diabetes mellitus involves the loss of function and/or absolute numbers of insulin-producing ß cells in pancreatic islets. Islet transplantation is currently being investigated as a potential cure, and advances in tissue engineering methods can be used to improve pancreatic islets survival and functionality. Transplanted islets experience anoikis, hypoxia, and inflammation-mediated immune response, leading to early damage and subsequent failure of the graft. Recent development in tissue engineering enables the use of decellularized organs as scaffolds for cell therapies. Decellularized pancreas could be a suitable scaffold as it can retain the native extracellular matrix and vasculature. In this study, mouse pancreata were decellularized by perfusion using 0.5% sodium dodecyl sulfate. Different characterizations revealed that the resulting matrix was free of cells and retained part of the pancreas extracellular matrix including the vasculature and its internal elastic basal lamina, the ducts with their basal membrane, and the glycosaminoglycan and collagen structures. Islets were infused into the ductal system of decellularized pancreata, and glucose-stimulated insulin secretion results confirmed their functionality after 48 hr. Also, recellularizing the decellularized pancreas with green fluorescent protein-tagged INS-1 cells and culturing the system over 120 days confirmed the biocompatibility and non-toxic nature of the scaffold. Green fluorescent protein-tagged INS-1 cells formed pseudoislets that were, over time, budding out of the decellularized pancreata. Decellularized pancreatic scaffolds seeded with endocrine pancreatic tissue could be a potential bioengineered organ for transplantation.

Tissue and organ decellularization in regenerative medicine.

The advancement and improvement in decellularization methods can be attributed to the increasing demand for tissues and organs for transplantation. Decellularized tissues and organs, which are free of cells and genetic materials while retaining the complex ultrastructure of the extracellular matrix (ECM), can serve as scaffolds to subsequently embed cells for transplantation. They have the potential to mimic the native physiology of the targeted anatomic site. ECM from different tissues and organs harvested from various sources have been applied. Many techniques are currently involved in the decellularization process, which come along with their own advantages and disadvantages. This review focuses on recent developments in decellularization methods, the importance and nature of detergents used for decellularization, as well as on the role of the ECM either as merely a physical support or as a scaffold in retaining and providing cues for cell survival, differentiation and homeostasis. In addition, application, status, and perspectives on commercialization of bioproducts derived from decellularized tissues and organs are addressed. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1494-1505, 2018.