This chapter covers the basic and essential aspects of "good practice" tissue culture. Core information on autoclaving and the sterilization procedures for tissue culture media and reagents are supplied. Descriptions of the classical methods of chemical and mechanical separation used for subculturing cells arc presented. We provide important directions on how to care for incubators, prevent and control bacterial, fungal, and mycoplasm contamination. Finally, tips on water purification and the care and cleaning of dissection instruments are outlined.

This chapter outlines a procedure for initiation of mouse neopallium (cerebral cortex and its underlying white matter) cell cultures and their development into nearly pure microglia cultures. The cytokine CSF-1 is required for the development, survival, and differentiation of microglia. In cultures, astroglia secrete CSF-1 into the medium (Hao et al. 1990, J. Neurosci. Res. 27, 314-323). Therefore, a simple protocol is described, originally developed by 12 (Hao et al. 1991, Int J. Dev. Neurosci. 9, 1-14), which utilizes CSF-1-secreting astroglia cultures for production of better than 99% pure cultures of microglia.

Introduction, Marston Manthorpe. Outgrowth Assays and Cortical Slice Cultures, Valerie Castellani and Jurgen Bolz. Microexplant Cultures of the Cerebellum, Bernard Rogister and Gustave Moonen. Aggregating Neural Cell Cultures, Paul Honegger and Florianne Monnet-Tschudi. Primary Cell Cultures of Peripheral and Central Neurons and Glia, Mary I. Johnson and Richard P. Bunge. Chick Spinal Somatic Motoneurons in Culture, Bernhard H. J. Juurlink. Neural Cells in Microcultures: In Vitro Biological Assays for Neuroactive Agents, Marston Manthorpe. Construction and Use of Compartmented Cultures, Robert B. Campenot. Astrocyte and Oligodendrocyte Cultures, Ruth Cole and Jean de Vellis. Generation of Mouse Astroglia and Microglia Cultures from Mouse Neopallium, Sergey Fedoroff and Arleen Richardson. Generation of Oligodendroblasts from Primary Cultures of Rat Neopallium, Bernhard H. J. Juurlink, Shawn K. Thorburne, and Richard M. Devon. Culture of Glial Cells from Human Brain Biopsies, Voon Wee Yong and Jack P. Antel. Colony Cultures, Sergey Fedoroff and Arleen Richardson. Herpes Simplex Virus Vectors for Gene Therapy of the Nervous System , Angela P. Dyer and Frank Tufaro. Immunostaining and Identification of Antigens, Colin J. Barnstable. Elimination of Cell Types from Mixed Neural Cell Cultures, Richard M. Devon. Quantification of Cells in Culture, Arleen Richardson and Sergey Fedoroff. Procedures for Subculturing Cells, Arleen Richardson and Sergey Fedoroff. Preparation of Tissue-Cultured Material for Electron Microscopical Observation, Richard M. Devon. Tips in Tissue Culture, Sergey Fedoroff and Arleen Richardson. Use of Dissecting Instruments in Tissue Culture, Arleen Richardson. Abbreviations and Definitions. Index.

The premise of colony culturing is the assumption that single viable cells can attach to the substratum, divide, and form a progeny of cells that constitute a cell colony or clone. Colony cultures can be initiated either from a disaggregated cell suspension made directly from animal tissue, or from primary or secondary cultures or cell lines. Colonies, especially ones initiated directly from tissues, are not all identical. They vary in the morphology of their cells, and in their size and compactness. The morphology and size of the colony depends on the kinds of cells plated, interactions between cells, the degree of their differentiation, the cell generation time, the composition of the medium, the type of substratum, and the physical conditions to which the cells are subjected.

Cells attach to the culture substratum with varying degrees of adherence. Some types of cells do not adhere at all and grow as cell suspensions in the medium. Such cells are referred to as nonadhering cells (nonanchorage-dependent cells). Cells that have to attach to the substratum to proliferate are referred to as adhering cells (anchorage-dependent cells).

We have shown previously that in tissue culture stellate astrocytes downregulate F-actin and actin binding proteins (ABPs) (Abd-El-Basset et al.: J Neurosci Res 30:1-17, 1991), whereas the reactive-like astrocytes upregulate their F-actin (Fedoroff et al.: Neuroscience 22:255-266, 1987). In the present study we report that in normal brain, as in tissue culture, neither F-actin nor alpha-actinin (an ABP) could be detected in stellate astrocytes. When a stab wound was made in brain, F-actin and alpha-actinin were upregulated in reactive astrocytes. We also demonstrated that reactive-like astrocytes in tissue culture express alpha-actinin, which has a "dotted" appearance when immunostained, and is colocalized with F-actin in a specific arrangement.

Cell enumeration using the hemocytometer is applicable when determining the number of cells in a suspension, and when the number of samples to be analyzed is relatively small. Hemocytometry is also useful for determining the proportion of singly dispersed cells in a suspension, and for estimating the frequency of viable cells.

Cells attach to the culture substratum with varying degrees of adherence (see Chapter 18 ). Cells that must attach to the substratum are referred to as "adhering cells" (anchorage-dependent cells). Cells that do not adhere and grow as cell suspensions in the medium are referred to as "nonadhering cells" (nonanchorage-dependent cells).

Colony cultures are based on the assumption that single viable cells can attach to the substratum, divide, and form a progeny of cells that constitute a cell colony. Colony cultures can be initiated either from the original disaggregated cell suspension made directly from animal tissue, or from primary or secondary cultures. When large numbers of viable cells are plated/cm2 of substratum, on culturing, a confluent layer of cells forms. When a small number of cells are plated/cm2 of substratum, then single, discrete cell colonies form. Colonies, especially ones initiated directly from tissues, are not necessarily uniform. The morphology and size of the colony depends on the kinds of cells plated, the degree of their differentiation, the cell generation time, the composition of the medium, the type of substratum, and the physical conditions. In practice, disaggregated tissue yields a variety of cells of varying degrees of maturity, resulting in heterogeneity in colony size and morphology. Colony cultures from secondary cultures usually have more uniform colonies.

Disaggregated neopallial cells from newborn C3H/HeJ mice were cloned in Grenier hybridoma tissue culture dishes, and culture wells that contained only one cell were marked. After 8–10 days of culturing, the cultures were fixed and double immunolabeled for microglia with Mac-1 antibody and for astroglia with antibody to GFAP. Each marked well containing a clone was identified as either a microglia, astroglia, mixed microglia–astroglia, or an unlabeled clone. The effect of LM cell line conditioned medium (LM-CM), which contains colony-stimulating factor-1, on the development of mixed microglia–astroglia clones was determined. Formation of mixed clones was dose dependent (P < 0.0001). We concluded that microglia and astroglia have a common progenitor cell and that the development of mixed clones is LM-CM dependent. J. Neurosci. Res. 50:477–486, 1997. © 1997 Wiley-Liss, Inc.

Microglia rapidly respond to Lipopolysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immune-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes, We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alanine-rich C kinase substrate) and MRP (MARCKS-related protein). Within 6 hours of addition, LPS induced a twofold increase in [H-3]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP, The differential effect of LPS on expression of MRP vs, MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS, TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription, CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS, Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1. (C) 1996 Wiley-Liss, Inc.

GliaVolume 14, Issue 4 p. 329-332 Short Communication Remembrances of amico bignami Luciano Angelucci, Luciano AngelucciSearch for more papers by this authorJerald J. Bernstein, Jerald J. BernsteinSearch for more papers by this authorElisabeth Bock, Elisabeth BockSearch for more papers by this authorSergey Fedoroff, Sergey FedoroffSearch for more papers by this authorLief Hertz, Lief HertzSearch for more papers by this authorEzw Giacobini, Ezw GiacobiniSearch for more papers by this authorJean Lauder, Jean LauderSearch for more papers by this authorGiulio Levi, Giulio LeviSearch for more papers by this authorRita Levi-Montalcini, Rita Levi-MontalciniSearch for more papers by this authorEnrico Mugnaini, Enrico MugnainiSearch for more papers by this authorMichael Norenberg, Michael NorenbergSearch for more papers by this authorRegino Perez-Polo, Regino Perez-PoloSearch for more papers by this authorAlain Privat, Alain PrivatSearch for more papers by this authorPaola S. Timiras, Paola S. TimirasSearch for more papers by this authorAntonia Vernadakis, Antonia VernadakisSearch for more papers by this author Luciano Angelucci, Luciano AngelucciSearch for more papers by this authorJerald J. Bernstein, Jerald J. BernsteinSearch for more papers by this authorElisabeth Bock, Elisabeth BockSearch for more papers by this authorSergey Fedoroff, Sergey FedoroffSearch for more papers by this authorLief Hertz, Lief HertzSearch for more papers by this authorEzw Giacobini, Ezw GiacobiniSearch for more papers by this authorJean Lauder, Jean LauderSearch for more papers by this authorGiulio Levi, Giulio LeviSearch for more papers by this authorRita Levi-Montalcini, Rita Levi-MontalciniSearch for more papers by this authorEnrico Mugnaini, Enrico MugnainiSearch for more papers by this authorMichael Norenberg, Michael NorenbergSearch for more papers by this authorRegino Perez-Polo, Regino Perez-PoloSearch for more papers by this authorAlain Privat, Alain PrivatSearch for more papers by this authorPaola S. Timiras, Paola S. TimirasSearch for more papers by this authorAntonia Vernadakis, Antonia VernadakisSearch for more papers by this author First published: August 1995 https://doi.org/10.1002/glia.440140409AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume14, Issue4August 1995Pages 329-332 RelatedInformation

The purpose of the studies was to determine how gross physical characteristics of cerebrospinal fluid (CSF) shunts and the cellular proliferative response to shunts contribute to shunt obstruction. Ventricular catheters with round holes, slots, and flanges were implanted into the lateral ventricles of rabbits for 4 weeks. All shunt designs were subject to ingrowth of tissue from the ventricle wall or choroid plexus. There were no qualitative or quantitative differences between normal and hydrocephalic rabbits. Astroglial cells from newborn mice were cultured on shunt catheters for 2 or 4 weeks. The growth of these cells was poor, probably because the cells cannot attach well to the silicone rubber substrate. Contact between the shunt catheter and vascularized brain tissue is the most important factor in the genesis of shunt obstruction.

In this chapter I will describe the differentiation of astrocytes of mouse neopallium in culture based mainly on our own observations. Cells were obtained from mouse embryos of different developmental stages, from the neural tube of early embryos to the brain of paranatal animals. The neopallium was dissected, then the cells were disaggregated and planted in culture in dilutions adjusted so as to be conducive to the formation of cell colonies rather than a monolayer of cells (22, 28). Such cultures select cells that (a) adhere to the substratum (in this case, the plastic of the tissue culture petri dishes, and (b) proliferate to form colonies. Approximately 25 per cent of colonies in our cultures start from single cells, thus forming true clones, others form from two or a few cells. Spatial relationships and interactions between cells in the colony determine its gross morphology and this is so constant that it identifies the various types of colony and, in turn, the cells composing them (23, 26).