Hello Juan Leal Doblado you can use some thickening agent like Fumed silica to get your desired results.

Abstract Rotating forms of suspension culture allow cells to aggregate into spheroids, prevent the de-differentiating influence of 2D culture, and, perhaps most importantly of all, provide physiologically relevant, in vivo levels of shear stress. Rotating suspension culture technology has not been widely implemented, in large part because the vessels are prohibitively expensive, labor-intensive to use, and are difficult to scale for industrial applications. Our solution addresses each of these challenges in a new vessel called a cell spinpod. These small 3.5 mL capacity vessels are constructed from injection-molded thermoplastic polymer components. They contain self-sealing axial silicone rubber ports, and fluoropolymer, breathable membranes. Here we report the two-fluid modeling of the flow and stresses in cell spinpods. Cell spinpods were used to demonstrate the effect of fluid shear stress on renal cell gene expression and cellular functions, particularly membrane and xenobiotic transporters, mitochondrial function, and myeloma light chain, cisplatin and doxorubicin, toxicity. During exposure to myeloma immunoglobulin light chains, rotation increased release of clinically validated nephrotoxicity cytokine markers in a toxin-specific pattern. Addition of cisplatin or doxorubicin nephrotoxins reversed the enhanced glucose and albumin uptake induced by fluid shear stress in rotating cell spinpod cultures. Cell spinpods are a simple, inexpensive, easily automated culture device that enhances cellular functions for in vitro studies of nephrotoxicity.

Introduction Cells throughout the body are exposed to flow shear stress as fluids stream past their membranes1,2. In the kidney, fluid from the blood is filtered by the glomerulus, and this ultrafiltrate flows past proximal tubule cells (PTC) that are responsible for reabsorbing water, sodium, glucose, amino acids, and diverse hormones and proteins. This is not a languid process—a healthy kidney generates over 100 mL of ultrafiltrate per minute and the PTC are responsible for reabsorbing 70% of this volume. During these processes, cells of the proximal tubule experience fluid shear stress. Calculations of the actual fluid shear stress in vivo is complicated by the varying dimensions of the tubules and varying composition of the ultrafiltrate as it moves down the tubule, but it is estimated that PTC are exposed to shear stress in the range of 0.04–0.50 dynes/cm21,3,4,5.Fluid shear stress has an important role in maintaining the differentiation of PTC2,6. Exposure to fluid shear stress in vitro increases PTC transport of proteins7,8,9,10,11,12, expression of microvilli10, and formation of tight junctions, with increased transepithelial electrical resistance13,14,15,16,17,18,19. Accordingly, in order to serve as meaningful and representative models of living kidneys, cultured PTC in vitro must be exposed to fluid shear stress in a quantifiable manner2,20.Generating fluid shear stress in vitro presents a variety of challenges (reviewed in2). Several technologies are available including orbital shakers, parallel plates, microfluidics with peristaltic pumps, and perfused hollow fibers8,13,21,22,23,24. Roller bottles, paddle stirrers, and shakers are inexpensive options that are quite suitable for microorganisms with rigid cell walls such as fungi, bacteria, and algae that can tolerate the high shear levels induced by turbulent flow and are relatively resistant to injury from impact against the vessel walls and paddles. However, mammalian cells require much gentler treatment to avoid cellular damage and to accurately mimic the shear levels they experience in vivo5,25. Microfluidic and parallel plate models can deliver low levels of fluid shear stress but are rarely practical for high throughput analysis due to high cost, complexity, and/or large vessel volumes2.Rotating suspension cultures, where cells float in a liquid milieu, have significant advantages for delivering physiologic levels of flow shear stress. Suspension culture technology has been modeled, validated experimentally, and partially standardized for routine use6,26,27. In a rotating suspension culture with no headspace (i.e. when the chamber is completely full of media), the cells and medium rotate as a cohesive mass in laminar flow with no turbulence1,28. The rotating wall vessel spins around a horizontal axis and the cells move in an annulus around the axis of rotation (Supplemental Video S1). As the cells tend to sediment under the influence of gravity, the rotation of the vessel, brings them back up into suspension. Adherence-dependent cells can still be grown in rotating suspension culture vessels after attachment to microcarrier beads.Rotating suspension cultures not only avoid the limitations of 2D cultures, but also expose free cells, cells on the surface of microcarrier beads, or cells in spheroids to continuous and uniform levels of fluid shear stress. In a rotating suspension culture with zero headspace, cells are exposed to ~ 0.04 to 0.50 dynes/cm2 of shear stress1,6,29,30,31,32. It is important to note that the rotation speed does not affect shear stress, but rather determines the diameter of the annulus through which the cells migrate. Hence, rotating suspension culture delivers the same shear on the same size and density cells in the same media every time regardless of rotation speed. This gives the investigator broad parameters for rotation speed and facilitates delivery of reproducible shear stress.Despite its advantages, rotating suspension culture has found limited application due to limitations of the currently available hardware2. For example, re-usable vessels have multiple components, each requiring sterilization by autoclaving at different temperatures, as well as requiring complex manual assembly in a sterile environment. During use, the vessels attach to spindle rotators that spin with great precision. However, the rotators are relatively expensive and only accommodate a few vessels at one time, constraining the design of experiments in which large numbers of replicates are desirable2. In the most common applications, rotating suspension cultures are used to generate a large batch of tissue spheroids that are then transferred to other (static) systems for experimentation; in this case the advantages of suspension culture are lost at the analysis step.There is a compelling need for small, affordable, simple-to-use suspension culture devices for studies with large numbers of replicates2,6. We report the development and utility of an inexpensive, simple to use, rotating suspension culture device, called a cell spinpod. Fluid shears in these injection-molded cell spinpods are illustrated with two-fluid modeling of flow and stresses. To demonstrate their utility, we used cell spinpods to deliver fluid shear stress to immortalized renal proximal tubule cells growing as a confluent monolayer on the surface of collagen carrier beads. The rotation of the cell spinpod keeps the beads in suspension and exposes the cells to low levels of fluid shear stress. With this model, we have demonstrated the effect of fluid shear stress on renal cell gene expression and cellular functions, particularly cell transporters, and the consequences of myeloma light chain, cisplatin, and doxorubicin toxicity. These studies demonstrate that immortalized renal cells exposed to fluid shear stress in cell spinpods can be a useful target for in vitro nephrotoxicity assays.Article

• Open Access
• Published: 29 October 2021

Cell spinpods are a simple inexpensive suspension culture device to deliver fluid shear stress to renal proximal tubular cells

• Timothy G. Hammond,
• Corey Nislow,
• Ivan C. Christov,
• Vecihi Batuman,
• Pranay P. Nagrani,
• Marjan Barazandeh,
• Rohit Upadhyay,
• Guri Giaever,
• Patricia L. Allen,
• Michael Armbruster,
• Allen Raymond &
• Holly H. Birdsall

Scientific Reports volume 11, Article number: 21296 (2021) Cite this article

Optimization and Stability of Cell–Polymer Hybrids Obtained by “Clicking” Synthetic Polymers to Metabolically Labeled Cell Surface Glycans

• Ruben M. F. Tomás
• and
• Matthew I. Gibson*

Cite this: Biomacromolecules 2019, 20, 7, 2726–2736

Publication Date:May 29, 2019

https://doi.org/10.1021/acs.biomac.9b00478

Copyright © 2019 American Chemical Society

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Biomacromolecules

Abstract

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Re-engineering of mammalian cell surfaces with polymers enables the introduction of functionality including imaging agents, drug cargoes or antibodies for cell-based therapies, without resorting to genetic techniques. Glycan metabolic labeling has been reported as a tool for engineering cell surface glycans with synthetic polymers through the installation of biorthogonal handles, such as azides. Quantitative assessment of this approach and the robustness of the engineered coatings has yet to be explored. Here, we graft poly(hydroxyethyl acrylamide) onto azido-labeled cell surface glycans using strain-promoted azide–alkyne “click” cycloaddition and, using a combination of flow cytometry and confocal microscopy, evaluate the various parameters controlling the outcome of this “grafting to” process. In all cases, homogeneous cell coatings were formed with >95% of the treated cells being covalently modified, superior to nonspecific “grafting to” approaches. Controllable grafting densities could be achieved through modulation of polymer chain length and/or concentration, with longer polymers having lower densities. Cell surface bound polymers were retained for at least 72 h, persisting through several mitotic divisions during this period. Furthermore, we postulate that glycan/membrane recycling is slowed by the steric bulk of the polymers, demonstrating robustness and stability even during normal biological processes. This cytocompatible, versatile and simple approach shows potential for re-engineering of cell surfaces with new functionality for future use in cell tracking or cell-based therapies.

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Cell-based therapies have expanded the repertoire of tools in modern medicine providing an arsenal of treatments in addition to conventional drugs or protein-based therapies. Chimeric antigen receptor (CAR)-T cell therapies have rapidly emerged as a potential treatment for multiple hematological malignancies through the introduction of cancer targeting receptors on T-cell surfaces. (1−3) However, viral vector transduction of T-cells, the process which randomly inserts the CAR transgenes into the genome, presents risks of insertional oncogenesis and gene silencing. Manufacturing practicality is also a concern due to production and quality control costs along with safety and ethical concerns. (4,5) Gene knock-in can mitigate some of these caveats; however, the efficiency of this procedure is 20%, compared to 68% for retroviruses, and off-target mutagenesis is still a concern. (6,7) Thus, unmodified cells require significant purification and separation. Genetic techniques are also not easily adaptable to introduce nonbiotic components such as drugs, tracking modalities, and non-natural amino acids. (8) Hence, there is considerable opportunity to explore synthetic tools to adapt cell surfaces both in medicine and bioscience.

Re-engineering of mammalian cell surfaces with synthetic polymers is emerging as an approach to enable rapid, simple and versatile chemical remodeling of cells to introduce non-natural functionality. Masking of cell surface antigens of erythrocytes and islet cells has been widely explored using polymer coatings to improve blood transfusions, (9−11) reduce malaria parasite binding, (12) and prevent the rejection of islet transplants. (13) Enhancement of islet transplants has also been achieved by controlling the immediate blood mediated inflammatory reaction through the addition of natural polymers, including heparin, (14) thrombomodulin (15) and urokinase. (16) Additionally, cell–cell interactions can be controlled through the addition of biotin/avidin interactions and hybridization of oligoDNA demonstrating potential future roles in inducing stem cell differentiation for cell-based therapies. (17)

Despite cell surface engineering with natural and synthetic polymers presenting many potential roles in cell-based therapies, challenges arise in the formation of effective, clinical-translatable methods. Amphiphilic polymers, such as alkylated poly(vinyl alcohol) or lipid-based poly(ethylene glycol), (18,19) allow direct passive insertion into the lipid bilayer membranes with minimal impact to membrane integrity and structures. Glycocalyx remodeling using lipid-based glycoconjugates has been demonstrated to be a powerful tool to introduce specific glycan epitopes to the cell-surface, allowing mediation of multiple biological processes. Bertozzi et al. demonstrated that introducing lipid-based Siglec-7 allows immunomodulation of the innate immune system, preventing natural-killer mediated killing of allogeneic and xenogenic primary cells. (20) Rat cortical neurons engineered with lipid-terminated chondroitin sulfate glycosaminoglycans (GAGs) have been used to enhance nerve growth factor-mediated signaling and promote neural outgrowth. (21) Additionally, neural differentiation of embryonic stem cells has been achieved through membrane incorporation of neoproteoglycans. (22) However, dissociation of lipid-based polymers occurs in under 24 h, with reports of a 50% drop within 8 h, (22) due to intrinsic membrane turnover processes. (21,23,24) Thus, biological applications are limited to this time frame and the nonspecific nature of lipid insertion methods.

Additional noncovalent approaches include electrostatic deposition of polycations onto the negatively charged membrane. These approaches dramatically and rapidly reduce cell viability (