Mammalian Cell Culture Conditions Research Articles

Thermostable bFGF Improves Cell Lifespan by Enhancing Cell Activity in the Long-Term Culture of Human Orbicularis Oculi Stem Cells.

Stem cells derived from human orbicularis oculi muscle (hOOM) are a valuable resource for cell therapy. However, when stem cells are continuously cultured, their abilities tend to deteriorate over time. One method to address this issue is to use basic fibroblast growth factor (bFGF) to maintain the stem cell functionality. The limitation is that bFGF is unstable under mammalian cell culture conditions with a half-life of only 8 hours, which poses a significant challenge to the production and maintenance of high-quality stem cells. In this study, we used thermostable bFGF (TS-bFGF) and demonstrated that hOOM-derived stem cells cultured with TS-bFGF exhibited superior proliferation, stem cell function, reduced reactive oxygen species, and cellular senescence delay effect compared to cells cultured with wild-type bFGF. Considering the pivotal role of stem cells in broad ranges of applications such as regenerative medicine and cultured meat, we anticipate that TS-bFGF, owing to its thermostability and long-lasting properties, will contribute significantly to the acquisition of high-quality stem cells.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.

Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

Embedding magnesium metallic particles in polycaprolactone nanofiber mesh improves applicability for biomedical applications

Magnesium (Mg) metal is of great interest in biomedical applications, especially in tissue engineering. Mg exhibits excellent in vivo biocompatibility, biodegradability and, during degradation, releases Mg ions (Mg2+) with the potential to improve tissue repair. We used electrospinning technology to incorporate Mg particles into nanofibers. Various ratios of Mg metal microparticles (<44 µm diameter) were incorporated into nanofiber polycaprolactone (PCL) meshes. Physicochemical properties of the meshes were analyzed by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), mechanical tensile testing, X-ray diffractometry and UV–VIS spectrophotometry. Biological properties of meshes were evaluated in vitro and in vivo. Under mammalian cell culture conditions, Mg-containing meshes released hydrogen gas and relative amounts of free Mg2+ that reflected the Mg/PCL ratios. All meshes were non-cytotoxic for 3T3 fibroblasts and PC-12 pheochromocytoma cells. In vivo implantation under the skin of mice for 3, 8 and 28 days showed that Mg-containing meshes were well vascularized, with improved measures of inflammation and healing compared to meshes without Mg. Evidence included an earlier appearance and infiltration of tissue repairing macrophages and, after 28 days, evidence of more mature tissue remodeling. Thus, these new composite nanofiber meshes have promising material properties that mitigated inflammatory tissue responses to PCL alone and improved tissue healing, thus providing a suitable matrix for use in clinically relevant tissue engineering applications. Statement of SignificanceThe biodegradable metal, magnesium, safely biodegrades in the body, releasing beneficial byproducts. To improve tissue delivery, magnesium metal particles were incorporated into electrospun nanofiber meshes composed of a biodegradable, biocompatible polymer, polycaprolactone (PCL). Magnesium addition, at several concentrations, did not alter PCL chemistry, but did alter physical properties. Under cell culture conditions, meshes released magnesium ions and hydrogen gas and were not cytotoxic for two cell types. After implantation in mice, the mesh with magnesium resulted in earlier appearance of M2-like, reparative macrophages and improved tissue healing versus mesh alone. This is in agreement with other studies showing beneficial effects of magnesium metal and provides a new type of scaffold material that will be useful in clinically relevant tissue engineering applications.

Induction of EGFP expression in Pichia pastoris during co-culture with human endothelial cell line

While Pichia pastoris has been developed into a versatile recombinant protein expression system, there are only few studies that have investigated the efficacious use of this yeast with human cells. In this study, we demonstrated that P. pastoris can be cultured under mammalian cell culture conditions and co-cultured with human endothelial cells. Co-cultures did not affect endothelial cell morphology or viability. Additionally, P. pastoris was induced to express enhanced green fluorescence protein when co-cultured with human endothelial cell line EA.hy926 under mammalian cell culture conditions. Our study provides data to support the use of P. pastoris as a vehicle for direct delivery of recombinant proteins to mammalian cells during co-culture.

Human Colostrum is a Rich Source of Cells with Stem Cell-Like Properties

Background and Objective: Stem cells have been isolated from various parts of the human body including bone marrow, umbilical cord blood and adipose tissue. Human breast milk as a source of stem cells is less studied with limited reports. In this pilot work, we have investigated the stem cell-like properties of cells derived from human colostrum using in vitro mechanistic assays.Material and Methods: Institutional Committee for Stem Cell Research’s approval and written informed consent from volunteering mothers were obtained prior to sample collection. Colostrum (n=4) samples were collected from healthy lactating mothers of neonates admitted in neonatal intensive care unit and postnatal wards. Cells from colostrum were cultured in Minimal Essential Medium – Alpha containing 10% Fetal Bovine Serum. These adherent growing cells were propagated in vitro and investigated for their differentiation potential into adipogenic and osteogenic lineages, anchorage independent spherogenesis and clonality under mammalian cell culture conditions. Results: Human colostrum was found to be richly cellular. Colostrum derived cells were capable of bi-lineage differentiation into osteogenic and adipogenic lineages within 7 days of induction. Colostrum derived cultures exhibited clonal growth and expansion under in vitro cell culture conditions. Colostrum derived cells also showed anchorage independent growth as spheres. Conclusion: This study provides compelling and pioneering evidence to cite that human colostrum is a rich source of stem cell-like cells. The study took into account in vitro functional properties of adherence, clonal expansion and bi-lineage differentiation. Validation with a larger cohort and multiple profiling of cells would further open newer vistas aimed at furthering the present understanding and value of colostrum stem cells in maternal-perinatal physiology.

Structural changes to resorbable calcium phosphate bioceramic aged in vitro

This work investigates the effect of mammalian cell culture conditions on 3D printed calcium phosphate scaffolds. The purpose of the studies presented was to characterise the changes in scaffold properties in physiologically relevant conditions. Differences in crystal morphologies were observed between foetal bovine serum-supplemented media and their unsupplemented analogues, but not for supplemented media containing tenocytes. Scaffold porosity was found to increase for all conditions studied, except for tenocyte-seeded scaffolds. The presence of tenocytes on the scaffold surface inhibited any increase in scaffold porosity in the presence of extracellular matrix secreted by the tenocytes. For acellular conditions the presence or absence of sera proteins strongly affected the rate of dissolution and the distribution of pore diameters within the scaffold. Exposure to high sera protein concentrations led to the development of significant numbers of sub-micron pores, which was otherwise not observed. The implication of these results for cell culture research employing calcium phosphate scaffolds is discussed.

Potential of boronic acid functionalized magnetic particles in the adsorption of human antibodies under mammalian cell culture conditions

In this work, we systematically evaluated the potential of using boronic acid functionalized magnetic particles in the capturing of human immunoglobulin G under typical mammalian cell culture conditions. For comparison, Protein A coated magnetic particles were also used. The binding pH was found to significantly influence the adsorption isotherms of boronic acid particles with the higher capacities (0.216 g IgG/g support) being observed at pH 7.4. Comparatively, this value was 0.109 g IgG/g support, for Protein A particles under the same conditions. Both particles revealed very fast adsorption kinetics with more than 70% of the maximum binding capacity being achieved in a few seconds. The effect of glucose and lactate, which are known to interact with boronic acid, was evaluated. For glucose, the binding capacity was significantly influenced by the pH and decreased as pH increased. At pH 9.5, a 70% lower binding capacity was observed for glucose concentrations as low as 0.5 g/l. The effect of lactate was less pronounced and almost pH independent reaching at most 20% decrease in binding capacity. Nevertheless, the effect of both molecules was always lower at pH 7.4. The optimization of the elution conditions enabled complete recovery of bound IgG from boronic acid particles using 50 mM Tris–HCl, 200 mM sorbitol, 200 mM NaCl at pH 8.5.

Combining Culture Techniques for Bartonella: the Best of Both Worlds

In this study we compared some common Bartonella culturing methodologies using four diverse species causing human illnesses. Based on a review of the literature, we focused on three major inconsistencies between protocols: base medium, cell coculture, and temperature. Our data showed that Bartonella tamiae demonstrated temperature-dependent growth limitations between common culturing conditions only 2°C apart. Additionally, growth of B. quintana was significantly enhanced by the presence of mammalian cell coculture under mammalian cell culture conditions; however, when the medium was modified to incorporate insect cell culture-based medium, coculturing with mammalian cells was no longer needed. In this study, we were able to overcome these temperature- and cell-dependent limitations and accommodate all of the strains tested by combining mammalian cell culture-based medium with insect cell culture-based medium.

A predictive high‐throughput scale‐down model of monoclonal antibody production in CHO cells

Multi-factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro-bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high-throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro-bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro-bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro-bioreactor system as a high-throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro-bioreactor results with those obtained in conventional 3 L bioreactors. Excellent agreement was observed between the micro-bioreactor and the bench-top bioreactor. The micro-bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P < 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro-scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods.

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.

Nanobacteria-Like Particles in Human Arthritic Synovial Fluids

We investigated the existence of nanosize particles in synovial fluids of rheumatoid arthritis and osteoarthritis patients. These specimens were cultured under mammalian cell culture conditions (37 degrees C; 5% CO2/95% air) for a long period. After about 2 months, many nanoparticles appeared and they gradually increased in number and in size. The nanobacteria-like particles exist in synovial fluids of arthritis patients. The possibility of their existence and pathogenesis in various diseases should be verified cautiously.

Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population.

The aim of this study was to detect, isolate and characterize the nanobacteria from human renal stones from a north Indian population, and to determine their role in biomineralization. Renal stones retrieved from the kidneys of 65 patients were processed and subjected to mammalian cell culture conditions. The isolated bacteria were examined using scanning (SEM) and transmission electron microscopy (TEM). They were characterized for the presence of DNA, proteins and antigenicity. The role of these bacteria in biomineralization was studied by using the (14)C-oxalate based calcium oxalate monohydrate (COM) crystallization assay. We observed the presence of apatite forming, ultrafilterable gram negative, coccoid microorganisms in 62% of the renal stones. SEM studies revealed 60-200 nm sized organisms with a distinct cell wall and a capsule. TEM images showed needle like apatite structures both within and surrounding them. They were heat sensitive, showed antibiotic resistance and accelerated COM crystallization. A potent signal corresponding to the presence of DNA was observed in demineralized nanobacterial cells by flow cytometry. The protein profile showed the presence of several peptide bands of which those of 18 kDa and 39kDa were prominent. Apatite forming nanosized bacteria are present in human renal stones and may play a role in the pathophysiology of renal stone formation by facilitating crystallization and biomineralization. However, further studies are required to establish the exact mechanism by which nanobacteria are involved in the causation of renal stones.