Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3 D) culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3 D cell models. Thus, the 3 D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3 D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting. In addition, the progress of employing 3 D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3 D cell culture systems were discussed.     

THE USE OF MILLIPORE FILTERS IN ULTRASTRUCTURAL STUDIES OF CELL CULTURES AND VIRUSES

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 x 10⁸ virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.     

Fish embryo cell cultures for derivation of stem cells and transgenic chimeras.

It has been demonstrated in mammalian systems that techniques using embryonal stem cells provide advantages over conventional injection of DNA into embryos for generation of transgenic animals. We employed cell culture approaches in an attempt to develop this technology for fish transgenesis. Using a trout embryo-derived mitogenic preparation in a specialized culture medium, we initiated replication of zebrafish blastula-derived cell cultures and expressed marker genes introduced into the cells by plasmid transfection. Reintroduction of cells from the cultures into blastula-stage embryos indicated that the cultured cells survived and may contribute to the developing organism.     

National hospital survey of anaerobic culture and susceptibility methods: III

To assess the current status of anaerobic bacteriology in the United States, we surveyed, by means of a questionnaire, 150 hospitals selected at random with bed capacities of 200-1000 and we received responses from 98 (65%). Ninety-eight percent processed anaerobic culture specimens with 21% sending them to reference laboratories. Almost all these hospitals processed blood and wound cultures for anaerobes and all used selective media for identification, including BBE (52%), LKV (77%), and PEA (53%) agars. All hospital laboratories attempted identification of blood culture isolates including 80% that attempted speciation. Wound cultures for anaerobic bacteria and sterile site cultures were also processed for anaerobes by almost all labs. Identification of B. fragilis group species to species level was performed only in 56% of labs always and 37% sometimes. Preformed enzyme kits were used by 66% of labs and 30% used special potency disks for identification. Susceptibility testing was performed in-house by 21% of hospital labs and sent out to reference labs an additional 20%. Susceptibility testing was attempted for all blood culture isolates by both hospital (21% of total labs) and reference laboratories, but only performed by 17% for sterile body site and 14% of the time for wound isolates. Etest was used most often followed by broth microdilution. No labs used the agar dilution or disk elution methods. The antimicrobials most often tested in hospital labs, predicated on the commercial panel used, were penicillin/ampicillin and clindamycin (15/18; 83%; 15% of total labs), metronidazole (16/18; 89%; 16% of total labs) and cefotetan and ampicillin/sulbactam (12/18; 67%; 12% of total labs), piperacillin/tazobactam (7/18; 39%; 7% of total labs), cefoxitin (9/18; 50%), imipenem (8/18; 44%), and chloramphenicol (6/18; 33%). Our current survey suggests that while many labs are processing anaerobic cultures, especially blood cultures, the identification of isolates and the performance of antimicrobial susceptibility testing of isolates are in disarray and in dire need of improvement.     

An Open Source Based High Content Screening Method for Cell Biology Laboratories Investigating Cell Spreading and Adhesion

Background

Adhesion dependent mechanisms are increasingly recognized to be important for a wide range of biological processes, diseases and therapeutics. This has led to a rising demand of pharmaceutical modulators. However, most currently available adhesion assays are time consuming and/or lack sensitivity and reproducibility or depend on specialized and expensive equipment often only available at screening facilities. Thus, rapid and economical high-content screening approaches are urgently needed.

Results

We established a fully open source high-content screening method for identifying modulators of adhesion. We successfully used this method to detect small molecules that are able to influence cell adhesion and cell spreading of Swiss-3T3 fibroblasts in general and/or specifically counteract Nogo-A-Δ20-induced inhibition of adhesion and cell spreading. The tricyclic anti-depressant clomipramine hydrochloride was shown to not only inhibit Nogo-A-Δ20-induced cell spreading inhibition in 3T3 fibroblasts but also to promote growth and counteract neurite outgrowth inhibition in highly purified primary neurons isolated from rat cerebellum.

Conclusions

We have developed and validated a high content screening approach that can be used in any ordinarily equipped cell biology laboratory employing exclusively freely available open-source software in order to find novel modulators of adhesion and cell spreading. The versatility and adjustability of the whole screening method will enable not only centers specialized in high-throughput screens but most importantly also labs not routinely employing screens in their daily work routine to investigate the effects of a wide range of different compounds or siRNAs on adhesion and adhesion-modulating molecules.     

Culture techniques for studies on the growth, development and reproduction of copepods and cladocerans under laboratory and in situ conditions: a review

SUMMARY. This review deals with culture techniques in use to estimate individual growth, development and reproduction of copepods and cladocerans in cultures. The conceptual aspects of growth, reproduction and feeding behaviour are briefly summarized. Marine copepods are included, because the techniques employed for the cultivation of these animals are often more advanced than those used for freshwater crustaceans. Most of the studies reviewed here were carried out under defined laboratory conditions, but a few were under field conditions, generally in small in situ enclosures. The culture methods described in this review are based on 272 studies published between 1910 and 1987. A large number of different culture systems are critically discussed in relation to culture conditions and culture techniques and the taxa used. Particular attention is given to the effect of food quality, and the problem of how to apply laboratory measurements to field populations so as to limit errors to the minimum. A more detailed summary is given in the section on 'Recommendations' at the end of the review.     

LOW-RESISTANCE JUNCTIONS BETWEEN CANCER CELLS IN VARIOUS SOLID TUMORS

Electrical coupling, which reveals the presence of specialized low-resistance intercellular junctions, has now been found in four types of tumors, Sarcoma 180, Novikoff hepatoma, and Morris hepatomas 3924-A and 7777. Coupled cancer cells were distinguished from coupled normal cells by intracellular marking techniques. Although the evidence suggests that coupling may be extensive in some cases, it is not possible to say that the coupling was normal. In particular, the results do not exclude less obvious defects in the specialized junctions, such as abnormal distribution or decreased permeability to molecules other than small inorganic ions. The results are discussed in relation to previous studies of coupling in Novikoff hepatomas and in cultures of S1801 and II cell lines.     

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 10⁸ cells/spermatogenic cell type from a starting population of 7-8 x 10⁸ cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.     

Plasmodium falciparum: Invasion and development in highly parasitized cultures

Summary We report that synchronized cultures ofPlasmodium falciparum with up to 40% parasitized cells can be obtained with the use of low, red blood cell suspensions and only daily replacement of culture medium. These cultures contained not only a reduced proportion of uninfected red cells but also of population of cells with brief and equal time of exposure to culture conditions. Such high parasitemias are desirable for studies of ring-staged parasites (for which enrichment techniques are not available) and late-staged parasites when the manipulations for enrichment are inappropriate or unsuccessful. This work was supported by Grant HL-21016 from the National Institutes of Health, Bethesda, MD Preliminary observations on this report were presented at the Poster session at the Red Cell 6th Ann Arbor Conference, Michigan, October, 1983.     

High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations-typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-microl aliquots of cultures with densities as low as 10(3) cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (alpha subclass), OM43 (beta subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.     

Recent advances in neural cell culture

Cells isolated from the avian and mammalian central and peripheral nervous system and cultured in vitro provide an opportunity to study in situ properties of neurons and glial cells under relatively simple and carefully controlled conditions. Since Harrison's success in maintaining in vitro embryonic frog spinal cord 80 years ago, neural tissue culture has developed into an important and versatile discipline of neuroscience. The techniques developed in the past fall into four broad classes: Explant cultures, which are explanted from specific neuroanatomic loci to substrates as small tissue fragments. Dissociated cell cultures, which involve the seeding of enzymatically or mechanically dispersed cells on various attachment substrates. Reaggregate cultures, which require re-association of dissociated cells into small aggregates. Purified cell populations, which are prepared by the isolation of different cell types by gradient centrifugation or other separation techniques. These cultures have been utilized in studying various aspects of brain development and function. In this review several areas of significant and stimulating development in neural cell culture have been documented. They include formulation of serum-free medium, effects of growth factors, utilization of cell type-specific markers, and isolation and culture of purified neuronal/glial cells.     

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents ³. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells ¹¹ are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period ⁴. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.     

Prevention of mycoplasma contamination in leukemia-lymphoma cell lines.

The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%.     

Method for the passaging of microcarrier cultures to a production scale for producing high titre Disabled Infectious Single Cycle-Herpes Simplex Virus Type-2

A complementary cell line CR2 is currently used to propagate the Disabled Infectious Single Cycle Herpes Simplex Virus Type 2 (DISC HSV-2) on a small laboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up our production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production times were factors which were taken into consideration whilst developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the small scale study for an envisaged manfacturing process.     

Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production

Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.     

Culturing marine bacteria – an essential prerequisite for biodiscovery

The potential for using marine microbes for biodiscovery is severely limited by the lack of laboratory cultures. It is a long-standing observation that standard microbiological techniques only isolate a very small proportion of the wide diversity of microbes that are known in natural environments from DNA sequences. A number of explanations are reviewed. The process of establishing laboratory cultures may destroy any cell-to-cell communication that occurs between organisms in the natural environment and that are vital for growth. Bacteria probably grow as consortia in the sea and reliance on other bacteria for essential nutrients and substrates is not possible with standard microbiological approaches. Such interactions should be considered when designing programmes for the isolation of marine microbes. The benefits of novel technologies for manipulating cells are reviewed, including single cell encapsulation in gel micro-droplets. Although novel technologies offer benefits for bringing previously uncultured microbes into laboratory culture, many useful bacteria can still be isolated using variations of plating techniques. Results are summarized for a study to culture bacteria from a long-term observatory station in the English Channel. Bacterial biodiversity in this assemblage has recently been characterized using high-throughput sequencing techniques. Although Alphaproteobacteria dominated the natural bacterial assemblage throughout the year, Gammaproteobacteria were the most frequent group isolated by plating techniques. The use of different gelling agents and the addition of ammonium to seawater-based agar did lead to the isolation of a higher proportion of Alphaproteobacteria. Variation in medium composition was also able to increase the recovery of other groups of particular interest for biodiscovery, such as Actinobacteria.     

A maximum-likelihood approach for building cell-type trees by lifting

Background

In cell differentiation, a less specialized cell differentiates into a more specialized one, even though all cells in one organism have (almost) the same genome. Epigenetic factors such as histone modifications are known to play a significant role in cell differentiation. We previously introduce cell-type trees to represent the differentiation of cells into more specialized types, a representation that partakes of both ontogeny and phylogeny.

Results

We propose a maximum-likelihood (ML) approach to build cell-type trees and show that this ML approach outperforms our earlier distance-based and parsimony-based approaches. We then study the reconstruction of ancestral cell types; since both ancestral and derived cell types can coexist in adult organisms, we propose a lifting algorithm to infer internal nodes. We present results on our lifting algorithm obtained both through simulations and on real datasets.

Conclusions

We show that our ML-based approach outperforms previously proposed techniques such as distance-based and parsimony-based methods. We show our lifting-based approach works well on both simulated and real data.