Anchorage-dependent Mammalian Cells Research Articles

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

Magnetoelastic sensors for real-time tracking of cell growth.

Magnetoelastic (ME) sensors, which can be remotely activated via magnetic fields, are an excellent choice for wireless monitoring of biological parameters due to their ability to be scaled into different sizes and have their surface functionalized for chemical or biological sensing. In this study, we present the application of a commercially available ME material (Metglas 2826 MB) to develop a sensor system that can monitor the attachment of anchorage-dependent mammalian cells in two-dimensionalin vitro cell cultures. Results obtained with the developed sensors and detection system correlated with microscopic image analysis of cell quantification, which showed a linear relationship between the sensor response and attached fibroblast cells on the sensor surface. It was also revealed that the developed ME sensor system is capable of providing temporal profiles of cell growth corresponding to different stages of cell attachment and proliferation in real-time.

Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.

Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.

Thermosensitive PHEMA microcarriers: ATRP synthesis, characterization, and usabilities in cell cultures

In this study, we developed a novel microcarrier to enhance the production of anchorage-dependent mammalian cells in large scale by preserving them from the effects of shear forces and to enhance their removal from the surface without using proteolytic enzymes and chelating agents. This ‘thermosensitive microcarrier’ was synthesized by the grafting thermoresponsive molecule, N-isopropylacrylamide (NIPAAm), to the crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) beads by surface-initiated atom transfer radical polymerization. NIPAAm was polymerized on bromine-activated beads’ surfaces to prepare PHEMA-g-PNIPAAm microcarriers. Then, they were chemically characterized by attenuated total reflectance Fourier transform infrared and electron spectroscopy for chemical analysis. Surface morphologies were further investigated by scanning electron microscope and atomic force microscopy techniques. The results of characterization studies confirmed that PNIPAAm was successfully grafted onto PHEMA beads by the means of atom transfer radical polymerization reaction. The cellular activities of PHEMA-g-PNIPAAm microcarriers were evaluated at static and dynamic culture conditions by using two types of cell lines with different morphology, i.e. L929 mouse fibroblasts and HS2 epithelial human keratinocytes. The microcarriers exhibited better cell adhesion and proliferation characteristics for both cell lines. Although their thermally induced cell detachment efficiencies are lower than that of trypsinization, thermally harvested cells preserved their surface morphologies and proliferation characteristics.

3,4‐dihydroxy‐L‐phenylalanine as a cell adhesion molecule in serum‐free cell culture

In this article, we examined the feasibility of using 3,4-dihydroxy-L-phenylalanine (DOPA) as a cell adhesion molecule in serum-free cultures of anchorage-dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X-ray photoelectron spectroscopy and energy-dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA-coated, fibronectin-coated, or no material-coated culture plates in serum-free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell-adhesion molecule for serum-free culture.

Regenerative medicine bioprocessing: Concentration and behavior of adherent cell suspensions and pastes

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage-dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5-15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 +/- 13%, demonstrating proof-of principle for achieving tissue-like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL-1444) used in this study was 22.4 microm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2-15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus.

Stress-sensitive nutrient consumption via steady and non-reversing dynamic shear in continuous-flow rotational bioreactors

Stress-sensitive biological response is simulated in a modified parallel-disk viscometer that implements steady and unidirectional dynamic shear under physiological conditions. Anchorage-dependent mammalian cells adhere to a protein coating on the surface of the rotating plate, receiving nutrients and oxygen from an aqueous medium that flows radially and tangentially, accompanied by transverse diffusion in the z-direction toward the active surface. This process is modeled as radial convection and axial diffusion with angular symmetry in cylindrical coordinates. The reaction/diffusion boundary condition on the surface of the rotating plate includes position-dependent stress-sensitive nutrient consumption via the zr- and zΘ-elements of the velocity gradient tensor at the cell/aqueous-medium interface. Linear transport laws in chemically reactive systems that obey Curie's theorem predict the existence of cross-phenomena between scalar reaction rates and the magnitude of the second-rank velocity gradient tensor, selecting only those elements of ∇ v experienced by anchorage-dependent cells that are bound to protein-active sites. Stress sensitivity via the formalism of irreversible thermodynamics introduces a zeroth-order contribution to heterogeneous reaction rates that must be quenched when nutrients, oxygen, chemically anchored cells, or vacant active protein sites are not present on the surface of the rotating plate. Computer simulations of nutrient consumption profiles via simple nth-order kinetics (i.e., n = 1,2) suggest that rotational bioreactor designs should consider stress-sensitivity when the shear-rate-based Damköhler number (i.e., ratio of the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the rotating plate) is greater than ≈ 25% of the stress-free Damköhler number. Rotational bioreactor simulations are presented for simple 1st-order, simple 2nd-order, and complex stress-free kinetics, where the latter includes a 4th-order rate expression that considers adsorption/desorption equilibria via the Fowler–Guggenheim modification of the Langmuir isotherm for receptor-mediated cell–protein binding, accompanied by the formation of receptor complexes. Dimensionless parameters are identified to obtain equivalent stress-free nutrient consumption in the exit streams of 2-dimensional creeping-flow rotational bioreactors and 1-dimensional laminar-flow tubular bioreactors. Modulated rotation of the active plate at physiological frequencies mimics pulsatile cardiovascular flow and demonstrates that these rotational bioreactors must operate above the critical stress-sensitive Damköhler number, identified under steady shear conditions, before dynamic shear has a distinguishable effect on bioreactor performance.

Pressure-sensitive nutrient consumption via dynamic normal stress in rotational bioreactors

Pressure-sensitive biological response is simulated in “rotating-cup” bioreactors with unidirectional modulations in compressive stress at the cylindrical wall that stimulate bone-tissue growth. Anchorage-dependent mammalian cells (i) adhere to a protein coating, (ii) receive nutrients and oxygen from an aqueous medium via radial diffusion toward the active surface, and (iii) respond to physiological modulations in centrifual-force-induced fluid pressure at the cell/aqueous-medium interface. This process is modeled by the classic diffusion equation (i.e., Fick's second law), with a time-dependent reaction/diffusion boundary condition at the wall. Non-reversing angular velocity modulations resemble pulsations at physiological frequencies. Computer simulations of nutrient consumption profiles suggest that rotational bioreactor designs should consider the effects of normal stress when the pressure-sensitive Damköhler number (i.e., ratio of the pressure-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the cylindrical wall), evaluated under steady rotation, is greater than ≈ 10–20% of the stress-free Damköhler number (i.e., β 0,1st- order = 0.025) for simple 1st-order stress-free kinetics, and ≈ 1% of the stress-free Damköhler number (i.e., β 0,2nd- order = 0.40) for complex 2nd-order stress-free nutrient consumption. When the peak-to-peak amplitude of angular velocity modulations of the cylindrical wall is the same as or larger than the angular velocity for steady rotation, the effect of non-reversing centrifugal-force-induced dynamic normal stress in rotational bioreactors, superimposed on steady rotation, can be significant when one is below the critical value of the pressure-sensitive Damköhler number that has been identified under steady rotation.

A gel-free 3D microfluidic cell culture system

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Segregated Mathematical Model for Growth of Anchorage-Dependent MDCK Cells in Microcarrier Culture

To describe the growth behavior of anchorage-dependent mammalian cells in microcarrier systems, various approaches comprising deterministic and stochastic single cell models as well as automaton-based models have been presented in the past. The growth restriction of these often contact-inhibited cells by spatial effects is described at levels with different complexity but for the most part not taking into account their metabolic background. Compared to suspension cell lines these cells have a comparatively long lag phase required for attachment and start of proliferation on the microcarrier. After an initial phase of exponential growth only a moderate specific growth rate is achieved due to restrictions in space available for cell growth, limiting medium components, and accumulation of growth inhibitors. Here, a basic deterministic unstructured segregated cell model for growth of Madin Darby Canine Kidney (MDCK) cells used in influenza vaccine production is described. Four classes of cells are considered: cells on microcarriers, cells in suspension, dead cells, and lysed cells. Based on experimental data, cell attachment and detachment is taken explicitly into account. The model allows simulation of the overall growth behavior in microcarrier culture, including the lag phase. In addition, it describes the time course of uptake and release of key metabolites and the identification of parameters relevant for the design and optimization of vaccine manufacturing processes.

The impact of process stress on suspended anchorage‐dependent mammalian cells as an indicator of likely challenges for regenerative medicines

To quantify the engineering shear constraint on processing, the effect of capillary shear stress (pipe flow) on suspended anchorage-dependent mammalian cells has been investigated. Exposure of cultured rat aortic smooth muscle cells to repeated capillary shear stress (2-120 N m(-2)) causes a decrease in total number of cells, number of intact cells and number of cells able to grow. The optimum wall shear stress for cell survival was found to be 10-50 N m(-2) (flowrate 4-20 mL/min, I.D. 0.45 mm). Cell populations which are able to grow after exposure to shear stress do not exhibit reduced growth rate or altered metabolism.

Optimal design of metabolic flux analysis experiments for anchorage‐dependent mammalian cells using a cellular automaton model

Metabolic flux analysis (MFA) is widely used to quantify metabolic pathway activity. Typical applications involve isotopically labeled substrates, which require both metabolic and isotopic steady states for simplified data analysis. For bacterial systems, these steady states are readily achieved in chemostat cultures. However, mammalian cells are often anchorage dependent and experiments are typically conducted in batch or fed-batch systems, such as tissue culture dishes or microcarrier-containing bioreactors. Surface adherence may cause deviations from exponential growth, resulting in metabolically heterogeneous populations and a varying number of cellular "nearest neighbors" that may affect the observed metabolism. Here, we discuss different growth models suitable for deconvoluting these effects and their application to the design and optimization of MFA experiments employing surface-adherent mammalian cells. We describe a stochastic two-dimensional (2D) cellular automaton model, with empirical descriptions of cell number and non-growing cell fraction, suitable for easy application to most anchorage-dependent mammalian cell cultures. Model utility was verified by studying the impact of contact inhibition on the growth rate, specific extracellular flux rates, and isotopic labeling in lactate for MCF7 cells, a commonly studied breast cancer cell line. The model successfully defined the time over which exponential growth and a metabolically homogeneous growing cell population could be assumed. The cellular automaton model developed is shown to be a useful tool in designing optimal MFA experiments.

Engineering microenvironment for expansion of sensitive anchorage-dependent mammalian cells

Tissue engineering involves ex vivo seeding of anchorage-dependent mammalian cells onto scaffolds, or transplanting cells in vivo. The cell expansion currently requires repeated cell detachment from solid substrata by enzymatic, chemical or mechanical means. The report here presents a high yield three-dimensional culture and harvest system circumventing the conventional detachment requirements. Cells mixed with dilute cationic collagen were microencapsulated within an ultra-thin shell of synthetic polymers. The cationic collagen could rapidly form a conformal layer of collagen fibers around cells to support cell proliferation and functions. The collagen could be readily removed from cells with a buffer rinse after harvesting from the fragile microcapsules. The cells harvested from this system demonstrate improved attachment, morphology and functions over conventionally cultured cells, upon binding to ligand-conjugated polymer surfaces. The harvested cells can be re-encapsulated and allowed to proliferate again, or used immediately in applications.

Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation.

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and 'inverse transition cycling'. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.

Electrotransfection of anchorage-dependent mammalian cells

Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing.

REVIEW- Progress in Concept and Practice of Growing Anchorage Dependent Mammalian Cells in 3D

REVIEW- Progress in Concept and Practice of Growing Anchorage Dependent Mammalian Cells in 3D

Review-progress in concept and practice of growing anchorage-dependent mammalian cells in three dimension.

Tissue culture has played a major role in the rapid advances made in medical science in the past 50 yr. The full potential of the technique, however, is limited by the fact that growth of cells is usually restricted to a monolayer accompanied by major decreases in many of their tissue-specific functions. This has been shown to be due, in large part, to the inadequate oxygenation of cells growing in tissue culture dishes. Studies that show that the high charge density and rigidity of the plastic and glass surfaces used for culture are also major factors limiting growth of cells to a monolayer, are reviewed. A new culture system has been developed in which cells are grown on substrata made using perfluorocarbons (PFCs) coated with collagen type 1 and other adhesive factors. Perfluorocarbons have a much higher solubility for oxygen than water and have been used as oxygen delivery systems to protect cells from hypoxia. These new PFC-based substrata can provide both the optimal level of oxygen cells need to maintain differentiated functions and the flexible and weaker type of adhesion that allows cells to round up, interact with each other, and when provided with adequate nutritional support, to grow in three dimension.

A cone disk atomizer for production of biocompatible magnetic microcapsules for culture of anchorage-dependent mammalian cells

A cone disk atomizer has been designed for preparation of gelatin/polystyrene magnetic microcapsules for use as microcarriers in anchorage-dependent mammalian cell culture. This apparatus facilitates continuous production of microcapsules and ignores surfactants or organic solvents in traditional emulsion. Compared with the flat-disk atomizer, the feeding flow is subjected to a normal pressure provided by the special cone-shaped disk and liquid slide is dramatically decreased in the cone-disk atomizer. A flow-rate-limitation is observed in both flat-disk atomization and cone-disk atomization. A 36% increase in flow-rate limitation was observed in cone-disk atomization than in flat-disk atomization indicating the potential of this method for large-scale preparations.

Study of the Growth Curve of Vero Cell with Statistical Counting Method Based on the SEM Images

Abstract For industrial-scale culture of anchorage-dependent mammalian cell, such as vero cell, growth on the microcarriers offers certain advantages. As previous research reported, growth process can be illustrated by a growth curve - cell density versus time, which shows the change of cell density through whole growth process including: first adhesion stage; spreading and division stage; and last confluent stage. The growth curves are controlled by the culture condition (culture medium, temperature, pH value, inoculation concentration, etc.) and space condition (kind of microcarrier, microcarrier size, and surface property, etc.). Therefore, the cell growth curve is not only the important basis for studying growth kinetics, but also for designing biological reactors. Traditionally, total cell concentration for the microcarrier cultures were determined using the crystal violet method, and the cell nuclei were counted with a hemacytometer. in this method, the cells attached on each microcarrier were trypsinized and dyed by crystal violet dye, so that the number of nuclei counted was statistically representative. in this work, the cells and microcarriers were examined by using an environmental SEM - KYKY1500. As a result, the moist cells and microcarriers in SEM images show their natural characteristics, because this environmental SEM permits biological sample to be observed directly in an environmental specimen chamber without normal preparation for biological sample. As for the normal preparation, it needs a long duration, e.g. 2 days normally, to do fixing, desiccating and gold-coating. in contrast, the operation for observation of the biological samples in this SEM is much simpler. in view of this, a statistical counting method based on the SEM images for determining cell concentration during microcarrier culture was developed.

Optimization of physical parameters for cell attachment and growth on macroporous microcarriers

The rates of cell attachment of the anchorage-dependent mammalian cell line Vero to the gelatin-based macroporous microcarrier Cultispher-G were determined under various conditions. An optimal rate of attachment (0.98 x 10(-2) min(-1)) occurred by an intermittent stirring regimen of 3 min stirring at 40 rpm per 33 min. This stirring regimen appeared to maximize cell-to-bead attachment and minimized cell aggregation which occurred at a broadly comparable rate.A further increase in the rate of cell-to-bead attachment occurred by preincubation of the microcarriers in serum-supplemented medium prior to cell inoculation in a serum-free medium. However, serum supplementation (>5%) was required for maximal cell growth. The pH of the medium had little effect on cell attachment over a broad range (pH 7.1-8.0). An initial cell/bead inoculum of 30 ensured an even distribution of cells on the available microcarriers with a low proportion of unoccupied beads.The rate of cell attachment to Cultispher-G was an order of magnitude lower than the determined value for the charged dextran microcarrier Cytodex-1, which was measured as 9.05 x 10(-2) min(-1). The optimal conditions for cell attachment were significantly different for the two bead types. Cell attachment to the electrostatic surface of the Cytodex-1 microcarriers was highly dependent on pH and serum supplementation. Cell aggregation during attachment to the Cytodex-1 microcarriers was minimal because of the higher rate of cell-microcarrier attachment.The porous nature of the Cultispher-G microcarriers allowed a maximum cell/bead loading of >1400, which was at least 3 times higher than equivalent loading of the cells on Cytodex-1. The Cultispher-G matrix also allowed the use of higher agitation rates (up to 100 rpm) in spinner flasks without affecting the cell growth rate or maximum cell density.