Basic techniques for mammalian cell tissue culture tritech. Basic protocols for zebrafish cell lines maintenance and transfection. Basic protocol 1 cmv u6 grna cas basic protocol 2 basic protocol 3 basic protocol 4 prepare cas9 and grna plasmids experimental timeline total. Basic pluripotent stem cell culture protocols ncbi bookshelf. Ecacc handbook fundamental techniques in cell culture sigma excellent volume of cell culture protocols from the european collection of cell cultures ecacc containing the following chapters. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Basic cell culture protocols methods in molecular biology in this fully revised edition of an established classic, expert researchers and clinicians describe in stepbystep detail updated techniques for the isolation and growth of major primary cell types, such as kidney proximal tubule cells, hepatocytes, keratinocytes, and cardiomyocytes. However, as long as a few basic guidelines are adopted this should not compromise the work. A manual of basic technique and specialized applications, seventh edition provides the most accessible and comprehensive introduction. The purpose of this chapter is to explain the basic principles of cell culture using the maintenance of a nonadherent cell line, the p815 mouse mastocytoma cell line, and adherent primary mouse embryonic fibroblasts mef as examples. Mammalian cell tissue culture techniques protocol abcam. After the first subculture, the primary cells start to become a.
Originally, the term is not limited to animal cells, but includes the in. Follow these simple rules to ensure healthy and reproducible cell cultures. The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture. A manual of basic technique and specialized applications posttitle from mediafire, rapishare, and mirror link. Many of these protocols feature helpful notes and safety information for practical application. Basic protocols for zebrafish cell lines article pdf available in methods in molecular biology 362. Totipotencya cell characteristic in which the potential for forming all the cell types in the adult organism are retained. Because of the continuing expansion of cell culture, and to keep the bulk of the book to a reasonable size, some specialized protocols are presented as supplementary material online. However, some of them are specific to tissue culture. Below are links to a number of different online protocols for the derivation and culture of human es and ips cells. Routine passaging of zebrafish cell cultures all cell culture work should be performed in a laminar flow tissue culture.
Basic cell culture protocols third edition volume 290 methodsmethods inin molecularmolecular biologybiology tmtm edited by cheryl d. Yeast high efficiency transformation dslam pdf pandey lab protocols. The basic protocol describes subculturing of a monolayer culture grown in. Note that cell culture conditions vary for each cell type. Surachai unchern laboratory practice on caco2 cell culture. Helgason department of cancer endocrinology, british columbia cancer agency, vancouver, british columbia, canada cindy l. B4 preparation of pluripotent stem cell culture medium supplies lsterilepipettetips 20 lsterilepipettetips 5mlsterileserologicalpipets 10mlsterileserologicalpipets 25mlsterileserologicalpipets.
Basic cell culture protocols methods in molecular biology. Cell culture basics handbook thermo fisher scientific. However, the successful culture and maintenance of human pluripotent stem cells hpscs in an. Many of them are common sense and apply to all laboratory areas. The protocol below describes how to subculture adherent and suspension cultured cells. We suggest that part a isolation of individual colonies be done in sequence between laboratory 1 and laboratory 3. Detailed protocols for most of these basic procedures are already published freshney, 2005 and. This includes media, glassware, and instruments, as well as the.
Cell culture protocols, hela and cho cells woods hole physiology course, 2006 hela cells olympus mircoscopy resource center. This is the most complete resource on the techniques, equipment, principles, and practices of animal cell culture. Crisprcas9directed genome editing of cultured cells. Since publication of the previous edition of this benchmark text, numerous groundbreaking advances have occurred in stem cell research, cloning, tissue. A more comprehensive reference on animal cell culture can be found in culture of animal cells. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Most cell lines will grow on culture flasks without the need for special matrixes etc. This book contains numerous useful protocols, covering light and electron microscopy, cell culture, cell separation, subcellular fractionation, organelle and membrane isolation, and the use of in vitro reassembly systems in cell biology. Basic techniques in mammalian cell tissue culture phelan. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell. In other words, it is an in vitro culture of plant cells or tissues on artificial. Cell culture protocols thermo fisher scientific in. General details of cell culturing and subculturing. Cell cultures are utilized in cytogenetic, biochemical, and molecular laboratories for diagnostic as well as research studies.
Suggested protocols for generation of spheroids from the most commonly used cancer cell lines using nunclon sphera cell culture plastics and gibco media. This book is a guide for the newcomer to all those areas which are currently basic to the performance of animal including human cell culture. Uptodate and highly practical, basic cell culture protocols, third edition, offers basic scientists and clinicianresearchers powerful tools to isolate, culture, and characterize the promising specialized cell types in demand today. Basic pluripotent stem cell culture protocol stembook. Cell culture protocols thermo fisher scientific tw. How to count and calculate the number of cells from a stock flask or culture dish. However, some cells, particularly primary cells, will require growth on special.
Inherent with successful manipulation of cell cultures is the basic understanding that everything that comes into contact with the cells must be sterile or noncontaminating. Stem cell basics last modified on april 08, 2015 about this document this primer on stem cells is intended for anyone who wishes to learn more about the biological properties of stem cells, the important questions about stem cells that are the focus of scientific research, and the potential use of stem cells in research and in treating disease. In addition to these protocols, we highly recommend stembook, which is publishing a growing list of methods to generate induced pluripotent stem cells and differentiate them into various. Unless they undergo an immortalization procedure, primary cells have a limited lifespan and usually reach senescence after 1020 passages. In this fully revised edition of an established classic, expert researchers and clinicians describe in stepbystep detail updated techniques for the isolation and growth of the major primary cell types, such as kidney proximal tubule cells, hepatocytes.
Cell culture guidelines the following is a general guideline for culturing of cell lines. Updating and significantly expanding upon the previous editions, basic cell culture protocols, fourth edition provides the novice cell culturist with sufficient information to perform the basic techniques, to ensure the health and identity of their cell lines, and to be able to isolate and culture specialized primary cell types. Tissue culturethe maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. Basic cell culture protocols third edition edited by cheryl d. Procedures for thawing, subculture, determination of cell numbers and viability. The following is a general guideline for culturing of cell lines. Cells in culture can be divided into three basic categories based on their shape and appearance i. The intent of this extensive volume is to generate a valuable resource containing clear methodologies pertinent to current areas of investigation, rather than. Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. The authors offer readily reproducible new methods for the differentiation of embryonic stem es cells into various hematopoietic cell types, for fetal thymic organ culture, and for the isolation and culture of specialized cell types, such as mammary progenitor cells, skeletal muscle myofibers, mesenchymal cells, neural stem cells, hematopoietic cells, stromal cell lines, and endothelial cells. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. Basic cell culture protocols, third edition edited by. Direct download links available pretitle culture of animal cells.
Stable isotope labeling with amino acids in cell culture silac protocol for quantitation of protein dynamics pdf 18o labeling trypsin digest protocol for relative quantitation by. This unit describes the basic skills required to maintain and preserve cell cultures. However, the successful culture and maintenance of human pluripotent stem cells hpscs in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of selfrenewal and. Basic protocols for zebrafish cell lines researchgate. The objective of this chapter is to highlight those principles and procedures that have particular relevance to the use of cell culture in tissue engineering. This is the sixth edition of the leading text in the basic methodology of cell culture, worldwide.