A simple apparatus for measuring cell settling velocity

Accurate cell settling velocity determination is critical for perfusion culture using a gravity settler for cell retention. We have developed a simple apparatus (a "settling column") for measuring settling velocity and have validated the procedure with 15-μm polystyrene particles with known physical properties. The measured settling velocity of the polystyrene particles is within 4% of the value obtained using the traditional Stokes' law approach. The settling velocities of three hybridoma cell lines were measured, resulting in up to twofold variation among cell lines, and the values decreased as the cell culture aged. The settling velocities of the nonviable cells were 33-50% less than the corresponding viable cells. The significant variation of settling velocities among cell populations and growth phases confirms the necessity of routine measurement of this property during long-term perfusion culture.     

A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

红豆杉细胞培养中紫杉醇高产细胞株的筛选及其稳定性分析

对中国红豆杉〔Taxus chinensis(Pilger)Rehd.〕、云南红豆杉(T. yunnanensis Cheng et L.K.Fu)和东北红豆杉(T. cuspidata Sieb.et Zucc.)不同部位的愈伤组织进行培养及分析比较。结果表明,中国红豆杉愈伤组织生长较快,其叶片诱导的愈伤组织紫杉醇含量较高,且紫杉醇含量与培养物的外观特征有明显相关性,颜色浅、块状或颗粒较明显的细胞团紫杉醇含量较高。运用细胞看护培养技术,从中国红豆杉愈伤组织中筛选出生长速率达0.52g.L-1.d-1、紫杉醇含量超过0.01%的细胞优株,经20次继代培养,其生长和紫杉醇含量均较稳定。     

Design and characterization of a new bioreactor for continuous ultra-slow uniaxial distraction of a three-dimensional scaffold-free stem cell culture

A computer controlled dynamic bioreactor for continuous ultra-slow uniaxial distraction of a scaffold-free three-dimensional (3D) mesenchymal stem cell pellet culture was designed to investigate the influence of stepless tensile strain on behavior of distinct primary cells like osteoblasts, chondroblasts, or stem cells without the influence of an artificial culture matrix. The main advantages of this device include the following capabilities: (1) Application of uniaxial ultra-slow stepless distraction within a range of 0.5-250 μm/h and real-time control of the distraction distance with high accuracy (mean error -3.4%); (2) tension strain can be applied on a 3D cell culture within a standard CO(2) -incubator without use of an artificial culture matrix; (3) possibility of histological investigation without loss of distraction; (4) feasibility of molecular analysis on RNA and protein level. This is the first report on a distraction device capable of applying continuous tensile strain to a scaffold-free 3D cell culture within physiological ranges of motion comparable to distraction ostegenesis in vivo. We expect the newly designed microdistraction device to increase our understanding on the regulatory mechanisms of mechanical strains on the metabolism of stem cells.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.     

Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures

Purkinje cells are vulnerable to a number of physical, chemical, and genetic insults during development and maturity. Normal development of these cells depends on the cell-cell interactions between granule and astroglial cell populations. Apoptotic death in Purkinje neurons had been shown to be associated with cell cycle activation, and new DNA synthesis is associated with Purkinje cell death in staggerer and lurcher mutant mice. Here using an in vitro organotypic slice culture model from 9 (P9) and 4 days (P4) old postnatal rats we show that the cyclin dependent kinase (cdk) inhibitors (roscovitine, olomoucine, and flavopiridol) protect the Purkinje cells from cell death. The results are more pronounced in the cerebellar sections from P4 rats. Analysis of Purkinje neurons in sections from P4 rats after 1 week of culturing showed that while there were very limited calbindin positive neurons in the untreated sections the cdk inhibitor treated sections had a notably higher number. Although treatment with cdk inhibitors inhibited Purkinje cell loss significantly, the morphology of these neurons was abnormal, with stunted dendrites and axons. Since the retinoblastoma protein (Rb) is the major pocket protein involved in determining the differentiated state of neurons we examined the effect of over-expressing Rb in the organotypic cultures. Rb overexpression significantly inhibited the Purkinje cell death and these neurons maintained their normal morphology. Thus our studies show that the cell death in Purkinje neurons observed in organotypic cultures is cell cycle dependent and the optimal survival requires Rb.     

Rat hepatocyte primary cell cultures

Summary The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of the cultures in various media was compared revealing that in complex media, particularly containing galactose, survival was improved. This study was supported by Contract No. N01-CP-55705 from the National Cancer Institute and Research Grant No. BC-133B from the American Cancer Society.     

Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

Kinetic characterization of baculovirus-induced cell death in insect cell cultures

The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (t(d)) and a first-order death phase characterized by a half-life (t(1/2)). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection (c) 1993 John Wiley & Sons, Inc.     

Spectroscopic methods and their applicability for high‐throughput characterization of mammalian cell cultures in automated cell culture systems

The number and use of automated cell culture systems for mammalian cell culture are steadily increasing. Automated cell culture systems require miniaturized analytics with a high throughput to obtain as much information as possible from single experiments. Standard analytics commonly used for conventional bioreactor samples cannot handle the high throughput and the low sample volumes. Spectroscopic methods provide a means of meeting this analytical requirement and afford fast and direct access to process information. In the first part of this review, UV/VIS, fluorescence, Raman, near‐infrared, and mid‐infrared spectroscopy are presented. In the second part of the review, these spectroscopic methods are evaluated in terms of their applicability in the new field of mammalian cell culture processes in automated cell culture systems. Unlike standard bioreactors, these automated systems have special requirements that apply to the use of spectroscopic methods. Therefore, they are compared with regard to cell culture automation, throughput, and required sample volume.     

Differential pulse polarography for monitoring cell cultures

The differential pulse polarography has been used on samples taken from the medium during the culture of chinese hamster ovary cells with a view to producing the active principle of an anti-cancer drug. An electrochemical signal at a potential of −1.8 V with respect to the saturated calomel electrode was correlated with the lactic acid concentration, the pH variation and the number of cells. The peaks observed in oxidation at potentials −0.35 V and −0.05 V were associated with the transformation of the cystine present in the nutrient medium into cysteine and provide an indication of the modification of the reducing power of the medium during culture. No correlation was found between the electrochemical behaviour and cell production.     

Treatment of mycoplasma contamination in a large panel of cell cultures

Summary Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the speciesMycoplasma arginini andM. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

A simple method for examining organotypic CNS cultures with nomarski optics

Summary This paper describes a method for examination of living organotypic cultures of CNS with Nomarski differential interference-contrast optics. Cultures grown in Maximow assemblies. which promote the best differentiation of the tissue but are optically faulty, are transferred for Nomarski observation to a simple sandwich chamber which combines the optical perfection of the usual sandwich chamber with the flexibility and safeguarding of sterility characteristic of the Maximow assembly. Thus cultures can be transferred repeatedly between their maintenance and observation chambers. In the resulting microscopic images, it is posible to visualize delicate unmyelinated fibers, myelinated cell bodies and other features which are normally impossible to demonstrate in living cultures as well as to improve the images of other structures such as large neuronal perikarya and myelinated axons. This work was supported in part by NIH Grant NS 11425     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures

Summary One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. We applied a rapid and efficient isoelectric focusing (IEF) technique for the routine analysis to detect interspecies contamination of cell cultures and for the identification of unknown animal cell lines. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between cell lines of human, murine, or other mammalian origin. By means of preformed agarose gels, standardized conditions and equipment, this technique is especially applicable for routine work and allows the analysis of a large number of unknown samples with reproducible results. One hundred seventy-seven cell lines which have been sent to the Department of Human and Animal Cell Cultures at the DSM (Deutsche Sammlung von Mikroorganismen and Zellkulturen) were analyzed for species authentication; only three cell lines were found not to be of the presumed species. Our study strongly emphasizes standardized IEF as an efficient and rapid method for routinely monitoring the authenticity of cell lines.     

Recent advances in organotypic tissue slice cultures for anticancer drug development

Organotypic tissue slice culture is established from animal or patient tissues and cultivated in an in vitro ecosystem. This technique has made countless contributions to anticancer drug development due to the vast number of advantages, such as the preservation of the cell repertoire and immune components, identification of invasive ability of tumors, toxicity determination of compounds, quick assessment of therapeutic efficacy, and high predictive performance of drug responses. Importantly, it serves as a reliable tool to stratify therapeutic responders from nonresponders and select the optimal standard-of-care treatment regimens for personalized medicine, which is expected to become a potent platform and even the gold standard for anticancer drug screening of individualization in the near future.     

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.