Human mesenchymal stromal cell adhesion and expansion on fluoropolymer surfaces modified with oxygen and nitrogen-rich plasma polymers.

Fluorinated ethylene propylene (FEP) vessels are of significant interest for therapeutic cell biomanufacturing applications due to their chemical inertness, hydrophobic surface, and high oxygen permeability. However, these properties also limit the adhesion and survival of anchorage-dependent cells. Here, we develop novel plasma polymer coatings to modify FEP surfaces, enhancing the adhesion and expansion of human mesenchymal stromal cells (hMSCs). Similar to commercially available tissue culture polystyrene vessels, oxygen-rich or nitrogen-rich surface chemistries can be achieved using this approach. While steam sterilization increased the roughness of the coatings and altered the surface chemistry, the overall wettability and oxygen or nitrogen-rich nature of the coatings were maintained. In the absence of proteins during initial cell attachment, cells adhered to surfaces even in the presence of chelators, whereas adhesion was abrogated with chelator in a protein-containing medium, suggesting that integrin-mediated adhesion predominates over physicochemical tethering in normal protein-containing cell seeding conditions. Albumin adsorption was more elevated on nitrogen-rich coatings compared to the oxygen-rich coatings, which was correlated with a higher extent of hMSC expansion after 3 days. Both the oxygen and nitrogen-rich coatings significantly improved hMSC adhesion and expansion compared to untreated FEP. FEP surfaces with nitrogen-rich coatings were practically equivalent to commercially available standard tissue culture-treated polystyrene surfaces in terms of hMSC yields. Plasma polymer coatings show significant promise in expanding the potential usage of FEP-based culture vessels for cell therapy applications.

Improving the 3D Printability of Sugar Glass to Engineer Sacrificial Vascular Templates.

A prominent obstacle in scaling up tissue engineering technologies for human applications is engineering an adequate supply of oxygen and nutrients throughout artificial tissues. Sugar glass has emerged as a promising 3D-printable, sacrificial material that can be used to embed perfusable networks within cell-laden matrices to improve mass transfer. To characterize and optimize a previously published sugar ink, we investigated the effects of sucrose, glucose, and dextran concentration on the glass transition temperature (Tg), printability, and stability of 3D-printed sugar glass constructs. We identified a sucrose ink formulation with a significantly higher Tg (40.0 ± 0.9°C) than the original formulation (sucrose-glucose blend, Tg = 26.2 ± 0.4°C), which demonstrated a pronounced improvement in printability, resistance to bending, and final print stability, all without changing dissolution kinetics and decomposition temperature. This formulation allowed printing of 10-cm-long horizontal cantilever filaments, which can enable the printing of complex vascular segments along the x-, y-, and z-axes without the need for supporting structures. Vascular templates with a single inlet and outlet branching into nine channels were 3D printed using the improved formulation and subsequently used to generate perfusable alginate constructs. The printed lattice showed high fidelity with respect to the input geometry, although with some channel deformation after alginate casting and gelation-likely due to alginate swelling. Compared with avascular controls, no significant acute cytotoxicity was noted when casting pancreatic beta cell-laden alginate constructs around improved ink filaments, whereas a significant decrease in cell viability was observed with the original ink. The improved formulation lends more flexibility to sugar glass 3D printing by facilitating the fabrication of larger, more complex, and more stable sacrificial networks. Rigorous characterization and optimization methods for improving sacrificial inks may facilitate the fabrication of functional cellular constructs for tissue engineering, cellular biology, and other biomedical applications.

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices.

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices' full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices' in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin-lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the "Oxygen Measurement Core" was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members' laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.

Bioaffinity-based surface immobilization of antibodies to capture endothelial colony-forming cells.

Maximizing the re-endothelialization of vascular implants such as prostheses or stents has the potential to significantly improve their long-term performance. Endothelial progenitor cell capture stents with surface-immobilized antibodies show significantly improved endothelialization in the clinic. However, most current antibody-based stent surface modification strategies rely on antibody adsorption or direct conjugation via amino or carboxyl groups which leads to poor control over antibody surface concentration and/or molecular orientation, and ultimately bioavailability for cell capture. Here, we assess the utility of a bioaffinity-based surface modification strategy to immobilize antibodies targeting endothelial cell surface antigens. A cysteine-tagged truncated protein G polypeptide containing three Fc-binding domains was conjugated onto aminated polystyrene substrates via a bi-functional linking arm, followed by antibody immobilization. Different IgG antibodies were successfully immobilized on the protein G-modified surfaces. Covalent grafting of the protein G polypeptide was more effective than surface adsorption in immobilizing antibodies at high density based on fluorophore-labeled secondary antibody detection, as well as endothelial colony-forming cell capture through anti-CD144 antibodies. This work presents a potential avenue for enhancing the performance of cell capture strategies by using covalent grafting of protein G polypeptides to immobilize IgG antibodies.

Pancreatic islets in bed with microvasculature-companions for life.

In Cell Stem Cell, Aghazadeh et al.1 show that human embryonic stem cell-derived pancreatic progenitors can reverse hyperglycemia for several weeks in streptozotocin-induced diabetic mice when co-transplanted with microvessel fragments into the subcutaneous space.

An in vitro Perfused Macroencapsulation Device to Study Hemocompatibility and Survival of Islet-Like Cell Clusters.

Transplantation of hydrogel-encapsulated pancreatic islets is a promising long-term treatment for type 1 diabetes that restores blood glucose regulation while providing graft immunoprotection. Most human-scale islet encapsulation devices that rely solely on diffusion fail to provide sufficient surface area to meet islet oxygen demands. Perfused macroencapsulation devices use blood flow to mitigate oxygen limitations but increase the complexity of blood-device interactions. Here we describe a human-scale in vitro perfusion system to study hemocompatibility and performance of islet-like cell clusters (ILCs) in alginate hydrogel. A cylindrical perfusion device was designed for multi-day culture without leakage, contamination, or flow occlusion. Rat blood perfusion was assessed for prothrombin time and international normalized ratio and demonstrated no significant change in clotting time. Ex vivo perfusion performed with rats showed patency of the device for over 100 min using Doppler ultrasound imaging. PET-CT imaging of the device successfully visualized metabolically active mouse insulinoma 6 ILCs. ILCs cultured for 7 days under static conditions exhibited abnormal morphology and increased activated caspase-3 staining when compared with the perfused device. These findings reinforce the need for convective transport in macroencapsulation strategies and offer a robust and versatile in vitro system to better inform preclinical design.

An immune regulatory 3D-printed alginate-pectin construct for immunoisolation of insulin producing ß-cells.

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co-block-polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1ß, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing ß-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment.

Accessible dynamic micropatterns in monolayer cultures via modified desktop xurography.

Micropatterned cell cultures provide an important tool to understand dynamic biological processes, but often require specialized equipment and expertise. Here we present subtractive bioscribing (SuBscribing), a readily accessible and inexpensive technique to generate dynamic micropatterns in biomaterial monolayers on-the-fly. We first describe our modifications to a commercially available desktop xurographer and demonstrate the utility and limits of this system in creating micropatterned cultures by mechanically scribing patterns into a brittle, non-adhesive biomaterial layer. Patterns are sufficiently small to influence cell morphology and orientation and can be extended to pattern large areas with complex reproducible shapes. We also demonstrate the use of this system as a dynamic patterning tool for cocultures. Finally, we use this technique to explore and improve upon the well-established epithelial scratch assay, and demonstrate that robotic control of the scratching tool can be used to create custom-shaped wounds in epithelial monolayers, and that the scribing direction leaves trace remnants of matrix molecules that may significantly affect conventional implementations of this common assay.

Developmentally-Inspired Biomimetic Culture Models to Produce Functional Islet-Like Cells From Pluripotent Precursors.

Insulin-producing beta cells sourced from pluripotent stem cells hold great potential as a virtually unlimited cell source to treat diabetes. Directed pancreatic differentiation protocols aim to mimic various stimuli present during embryonic development through sequential changes of in vitro culture conditions. This is commonly accomplished by the timed addition of soluble signaling factors, in conjunction with cell-handling steps such as the formation of 3D cell aggregates. Interestingly, when stem cells at the pancreatic progenitor stage are transplanted, they form functional insulin-producing cells, suggesting that in vivo microenvironmental cues promote beta cell specification. Among these cues, biophysical stimuli have only recently emerged in the context of optimizing pancreatic differentiation protocols. This review focuses on studies of cell-microenvironment interactions and their impact on differentiating pancreatic cells when considering cell signaling, cell-cell and cell-ECM interactions. We highlight the development of in vitro cell culture models that allow systematic studies of pancreatic cell mechanobiology in response to extracellular matrix proteins, biomechanical effects, soluble factor modulation of biomechanics, substrate stiffness, fluid flow and topography. Finally, we explore how these new mechanical insights could lead to novel pancreatic differentiation protocols that improve efficiency, maturity, and throughput.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags.

BACKGROUND: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. METHODS: Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. RESULTS: Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. CONCLUSIONS: Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Surface grafting of Fc-binding peptides as a simple platform to immobilize and identify antibodies that selectively capture circulating endothelial progenitor cells.

Antibody surface immobilization is a promising strategy to capture cells of interest from circulating fluids in vitro and in vivo. An application of particular interest in vascular interventions is to capture endothelial progenitor cells (EPCs) on the surface of stents to accelerate endothelialization. The clinical impact of EPC capture stents has been limited by the lack of efficient selective cell capture. Here, we describe a simple method to immobilize a variety of immunoglobulin G antibodies through their fragment crystallizable (Fc) regions via surface-conjugated RRGW peptides for cell capture applications. As an EPC capture model, peripheral blood endothelial colony-forming cells suspended in cell culture medium with up to 70% serum were captured by immobilized anti-CD144, anti-CD34 or anti-CD309 antibodies under laminar flow. The endothelial colony-forming cells were successfully enriched from a mixture with peripheral blood mononuclear cells using surfaces with anti-CD309 but not anti-CD45. This antibody immobilization approach holds great promise to engineer vascular biomaterials with improved EPC capture potential. The ease of immobilizing different antibodies using the same Fc-binding peptide surface grafting chemistry renders this platform suitable to screen antibodies that maximize cell capture efficiency and selectivity.

The importance and future of biochemical engineering.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

Atmospheric pressure cold plasma versus wet-chemical surface treatments for carboxyl functionalization of polylactic acid: A first step toward covalent immobilization of bioactive molecules.

The use of polylactic acid (PLA) has attracted growing interest, particularly in recent years, for biomedical applications because of its mechanical properties, biocompatibility, and biodegradability. Despite this, features such as surface hydrophobicity and the absence of suitable functional groups for covalent immobilization of bioactive molecules, make it challenging to endow PLA-based medical devices with additional features and thus broaden their range of applicability. In the present study, we demonstrate the suitability of atmospheric pressure dielectric barrier discharges operating in the Townsend regime as a promising alternative to other surface treatments, such as diazonium and alkali hydrolytic treatments, for carboxyl functionalization of PLA. Chemical changes in PLA surfaces are evaluated by contact angle measurements and by X-ray photoelectron spectroscopy while physical changes are investigated by scanning electron microscopy and atomic force microscopy. The amount of carboxyl groups generated on PLA surfaces is assessed by toluidine blue O assay and substantiated by grafting, through carboxyl groups, a fluorescent probe containing amino functionalities. All of the surface treatments have proven to be very effective in generating carboxylic groups on the PLA surface. Nevertheless, plasma treatment is shown to not degrade the PLA surface, in sharp contrast with diazonium and alkali hydrolytic treatments.

Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells.

Pluripotent stem cell (PSC)-derived insulin-producing cells are a promising cell source for diabetes cellular therapy. However, the efficiency of the multi-step process required to differentiate PSCs towards pancreatic beta cells is variable between cell lines, batches and even within cultures. In adherent pancreatic differentiation protocols, we observed spontaneous local clustering of cells expressing elevated nuclear expression of pancreatic endocrine transcription factors, PDX1 and NKX6.1. Since aggregation has previously been shown to promote downstream differentiation, this local clustering may contribute to the variability in differentiation efficiencies observed within and between cultures. We therefore hypothesized that controlling and directing the spontaneous clustering process would lead to more efficient and consistent induction of pancreatic endocrine fate. Micropatterning cells in adherent microwells prompted clustering, local cell density increases, and increased nuclear accumulation of PDX1 and NKX6.1. Improved differentiation profiles were associated with distinct filamentous actin architectures, suggesting a previously overlooked role for cell-driven morphogenetic changes in supporting pancreatic differentiation. This work demonstrates that confined differentiation in cell-adhesive micropatterns may provide a facile, scalable, and more reproducible manufacturing route to drive morphogenesis and produce well-differentiated pancreatic cell clusters.

Chitosan Microbeads Produced by One-Step Scalable Stirred Emulsification: A Promising Process for Cell Therapy Applications.

Cell microencapsulation is a promising approach to improve cell therapy outcomes by protecting injected cells from rapid dispersion and allowing bidirectional diffusion of nutrients, oxygen, and waste that promote cell survival in the target tissues. Here, we describe a simple and scalable emulsification method to encapsulate animal cells in chitosan microbeads using thermosensitive gel formulations without any chemical modification and cross-linker. The process consists of a water-in-oil emulsion where the aqueous phase droplets contain cells (L929 fibroblasts or human mesenchymal stromal cells), chitosan acidic solution and gelling agents (sodium hydrogen carbonate and phosphate buffer or beta-glycerophosphate). The oil temperature is maintained at 37 °C, allowing rapid physical gelation of the microbeads. Alginate beads prepared with the same method were used as a control. Microbeads with a diameter of 300-450 µm were successfully produced. Chitosan and alginate (2% w/v) microbeads presented similar rigidity in compression, but chitosan microbeads endured >80% strain without rupture, while alginate microbeads presented fragile breakage at <50% strain. High cell viability and metabolic activity were observed after up to 7 days in culture for encapsulated cells. Mesenchymal stromal cells encapsulated in chitosan microbeads released higher amounts of the vascular endothelial growth factor after 24 h compared to the cells encapsulated in manually cast macrogels. Moreover, microbeads were injectable through 23G needles without significant deformation or rupture. The emulsion-generated chitosan microbeads are a promising delivery vehicle for therapeutic cells because of their cytocompatibility, biodegradation, mechanical strength, and injectability. Clinical-scale encapsulation of therapeutic cells such as mesenchymal stromal cells in chitosan microbeads can readily be achieved using this simple and scalable emulsion-based process.

Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy.

Conventional cell culture surfaces typically consist of polystyrene, with or without surface modifications created through plasma treatment or protein/peptide coating strategies. Other polymers such as fluorinated ethylene propylene are increasingly being implemented in the design of closed cell culture vessels, for example to facilitate the production of cells for cancer immunotherapy. Cultured cells are sensitive to culture vessel material changes through different mechanisms including cell-surface interactions, which are in turn dependent on the amount, type, and conformation of proteins adsorbed on the surface. Here, we investigate the protein deposition from cell culture medium onto untreated polystyrene and fluoropolymer surfaces using quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both of these non-polar surfaces showed comparable protein deposition kinetics and resulted in similar mechanical and topographical film properties. At protein concentrations found in typical serum-free media used to culture dendritic cells, two deposition phases can be observed. The protein layers form within the first few minutes of contact with the cell culture medium and likely consist almost exclusively of albumin. It is indicated that initial protein film formation will be completed prior to cell settling and initial cell contact will be established with the secondary protein layer. The structural properties of the protein film surface will strongly depend on the albumin concentration in the medium and presumably be less affected by the chemical composition of the cell culture surface.

Isolating and expanding endothelial progenitor cells from peripheral blood on peptide-functionalized polystyrene surfaces.

The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces. Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion.

Development of a microchannel emulsification process for pancreatic beta cell encapsulation.

In this study, we developed a high-throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5-2.5% alginate solution with cells and CaCO3 across a 1-mm thick polytetrafluoroethylene plate with 700 × 200 µm rectangular straight-through channels. Alginate beads ranging from 1.5-3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical-scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.

Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell-based immunotherapies.

In recent years, cell-based therapies targeting the immune system have emerged as promising strategies for cancer treatment. This review summarizes manufacturing challenges related to production of antigen presenting cells as a patient-tailored cancer therapy. Understanding cell-material interactions is essential because in vitro cell culture manipulations to obtain mature antigen-producing cells can significantly alter their in vivo performance. Traditional antigen-producing cell culture protocols often rely on cell adhesion to surface-treated hydrophilic polystyrene flasks. More recent commercial and investigational cancer immunotherapy products were manufactured using suspension cell culture in closed hydrophobic fluoropolymer bags. The shift to closed cell culture systems can decrease risks of contamination by individual operators, as well as facilitate scale-up and automation. Selecting closed cell culture bags over traditional open culture systems entails different handling procedures and processing controls, which can affect product quality. Changes in culture vessels also entail changes in vessel materials and geometry, which may alter the cell microenvironment and resulting cell fate decisions. Strategically designed culture systems will pave the way for the generation of more sophisticated and highly potent cell-based cancer vaccines. As an increasing number of cell-based therapies enter the clinic, the selection of appropriate cell culture vessels and materials becomes a critical consideration that can impact the therapeutic efficacy of the product, and hence clinical outcomes and patient quality of life.

Dynamics of Endothelial Cell Responses to Laminar Shear Stress on Surfaces Functionalized with Fibronectin-Derived Peptides.

Surface endothelialization could improve the long-term performance of vascular grafts and stents. We previously demonstrated that aerosol-generated fibronectin-derived peptide micropatterns consisting of GRGDS spots over a WQPPRARI background increase endothelial cell yields in static cultures. We developed a novel fluorophore-tagged RGD peptide (RGD-TAMRA) to visualize cell-surface interactions in real-time. Here, we studied the dynamics of endothelial cell response to laminar flow on these peptide-functionalized surfaces. Endothelial cells were exposed to 22 dyn/cm2 wall shear stress while acquiring time-lapse images. Cell surface coverage and cell alignment were quantified by undecimated wavelet transform multivariate image analysis. Similar to gelatin-coated surfaces, surfaces with uniform RGD-TAMRA distribution led to cell retention and rapid cell alignment (∼63% of the final cell alignment was reached within 1.5 h), contrary to the micropatterned surfaces. The RGD-TAMRA peptide is a promising candidate for endothelial cell retention under flow, and the spray-based micropatterned surfaces are more promising for static cultures.