Animal Cell Culture

Essential Methods

John M. Davis, John M. Davis

• Éditeur: Wiley
• Année: 2011
• ISBN:
  9780470975633
• Édition: 1
• Sujet: Biowissenschaften
• Sous-sujet: Zellbiologie

This is a comprehensive research guide that describes both the key new techniques and more established methods. Every chapter discusses the merits and limitations of the various approaches and then provides selected tried-and-tested protocols, as well as a plethora of good practical advice, for immediate use at the bench. It presents the most accessible and comprehensive introduction available to the culture and experimental manipulation of animal cells. Detailed protocols for a wide variety of methods provide the core of each chapter, making new methodology easily accessible.
This book is an essential laboratory manual for all undergraduates and graduates about to embark on a cell culture project. It is a book which both experienced researchers and those new to the field will find invaluable.

1

The Cell Culture Laboratory

Sue Clarke1 and Janette Dillon2

1ImmunoBiology Limited, Babraham Research Campus, Babraham, Cambridge, UK

2Medlmmune, Granta Park, Cambridge, UK

1.1 Introduction

Cell culture dates back to the early twentieth century (Table 1.1) by which time some progress had already been made in cryopreservation, the long-term storage of mammalian cells in a viable state.

Table 1.1 The early years of cell and tissue culture.

Late nineteenth century – Methods established for the cryopreservation of semen for the selective breeding of livestock for the farming industry

1907 – Ross Harrison [1] published experiments showing frog embryo nerve fibre growth in vitro

1912 – Alexis Carrel [2] cultured connective tissue cells for extended periods and showed heart muscle tissue contractility over 2–3 months

1948 – Katherine Sanford et al. [3] were the first to clone – from L-cells

1952 – George Gey et al. [4] established HeLa from a cervical carcinoma – the first human cell line

1954 – Abercrombie and Heaysman [5] observed contact inhibition between fibroblasts – the beginnings of quantitative cell culture experimentation

1955 – Harry Eagle [6] and, later, others developed defined media and described attachment factors and feeder layers

1961 – Hayflick and Moorhead [7] described the finite lifespan of normal human diploid cells

1962 – Buonassisi et al. [8] published methods for maintaining differentiated cells (of tumour origin)

1968 – David Yaffe [9] studied the differentiation of normal myoblasts

The laboratory process of cell culture allows cells to be manipulated and investigated for a number of applications, including:

• studies of cell function, for example metabolism;
• testing of the effects of chemical compounds on specific cell types;
• cell engineering to generate artificial tissues;
• large-scale synthesis of biologicals such as therapeutic proteins and viruses.

The pioneering work of Ross Harrison in 1907 [1] demonstrated that culturing tissue in vitro (in glass) not only kept cells alive, but enabled them to grow as they would in vivo (in life). However, the early development of cell culture technology was hindered by issues of microbial contamination. The growth rate of animal cells is relatively slow compared with that of bacteria. Whereas bacteria can double every 30 minutes or so, animal cells require around 24 hours. This makes animal cell cultures vulnerable to contamination, as a small number of bacteria soon outgrow a larger population of animal cells. However, tissue culture became established as a routine laboratory method by the 1950s with the advent of defined culture media devised by Eagle and others. The discovery of antibiotics by Fleming was of course another major milestone that facilitated prolonged cell culture by reducing contamination issues.

In the 1940s and 1950s major epidemics of (among others) polio, malaria, typhus, dengue and yellow fever stimulated efforts to develop effective vaccines. It was shown in 1949 that poliovirus could be grown in cultures of human cells, and this became one of the first commercial ‘large-scale’ vaccine products of cultured mammalian cells. By the 1970s methods were being developed for the growth of specialized cell types in chemically defined media. Gordon Sato and his colleagues [10] published a series of papers on the requirements of different cell types for attachment factors such as high molecular weight glycoproteins, and hormones such as the insulin-like growth factors. These early formulations and mixtures of supplements still form the basis of many basal and serum-free media used today (see Chapters 4 and 5).

Recombinant DNA technology (also known as genetic engineering) was developed in the 1970s and it soon became apparent that large complex proteins of therapeutic value could be produced from animal cells. Another milestone came in 1975 with the production of hybridomas by Köhler and Milstein [11]. These cell lines, formed by the fusion of a normal antibody-producing cell with an immortal cancer (myeloma) cell, are each capable of the continuous production of antibody molecules with (in the modern embodiment of the technology) a single, unique amino acid sequence. By 2007, the centenary year of tissue culture, such monoclonal antibodies were being commercially produced in multi-kilogram quantities.

Large-scale culture applications have led to the manufacture of automated equipment, and today's high-end cell culture robots are able to harvest, determine cell viability and perform all liquid handling. The CellmateTM, for example, is a fully automated system for T-flasks and roller bottles [12] that was first produced for Celltech's manufacturing operations, and which has since been used in vaccine production. The latest version includes software to support validation if it is used in processes requiring compliance with regulations such as 21CFR Part 11 (see Chapter 11, Section 11.3.5). Also on the market are automatic cell culture devices that handle the smaller volumes used by high-throughput laboratories. This recognizes the growing importance of cell-based assays, particularly in the pharmaceutical industry. The CelloTM is an automated system for the culture of adherent and non-adherent mammalian cells in 6-, 24-, 96- and 384-well plates for the selection of optimal clones and cell lines. It automates operations from seeding through expansion and subculturing, and thereby decreases the time required for cell line development.

In the biomedical field cultured cells are already used routinely for a variety of applications, for example Genzyme's Epicel® (cultured epidermal keratinocyte autografts) for burns patients and Carticel® (cultured autologous chondrocytes) for cartilage repair, as well as at in vitro fertilization (IVF) clinics where the zygote is cultured – usually for a few days – prior to implantation in the mother's uterus. Stem cell research is another cell culture application that holds huge promise for the future, especially now specific cell programming is possible. Although much stem cell research used to depend on the use of embryonic stem cells (obtained from early-stage embryos,) scientists can, at least in some cases, now change differentiated somatic cells into stem cells (iPS – induced pluripotent stem cells) using genetically engineered viruses, mRNA or purified proteins, thus avoiding the ethical issues surrounding the use of embryos as a source of cells. These iPS cells are similar to classic embryonic stem cells in many of their molecular and functional features, and are capable of differentiating into various cell types, such as beating cardiac muscle cells, neurons and pancreatic cells [13]. Stem cells can potentially be used to replace or repair damaged cells, and promise to drastically change the treatment of conditions such as cancer, Alzheimer's and Parkinson's diseases, and even paralysis.

1.2 Methods and approaches

1.2.1 Cell culture laboratory design

When planning a new cell culture facility, it is important to clearly identify the type of work anticipated within the laboratory, as much will depend on the nature and scale of the culture to be performed. For any design, the access doors need to be large enough for the passage of any major equipment (an obvious point, but sadly one often overlooked). Even routine small-scale work, such as much of the tissue culture undertaken in healthcare, biotechnology and academia, has varied needs that require careful consideration in the planning stage.

Certain types of laboratory involved in highly specific work – such as IVF laboratories, environments dedicated to the production of biopharmaceuticals under Good Manufacturing Practice (GMP) conditions, or work with Hazard Group 4 pathogens (biological agents that are likely to cause severe human disease and pose a serious hazard to laboratory workers, are likely to spread to the community and for which there is usually no effective prophylaxis or treatment available) [14] – are not dealt with here. They require expert help for laboratory design because of the need to comply with stringent legislation and/or the highly significant associated health and safety risks.

Some of the questions that need to be answered before commencing the design of a laboratory are set out below. This list of questions is by no means exhaustive.

1.2.1.1 Health and safety implications

• What is the highest Hazard Group of material to be handled [15]?
• Will all work need to be carried out at the related containment level [14]? If not, what facilities are required at what level? (The lower the containment level, the less onerous and expensive it is to build, equip, run and work in the laboratory.)
• Will genetically modified organisms (GMOs) be used? (Note that, in the UK, any work with (or storage of) GMOs falls under the Genetically Modified Organisms (Contained Use) Regulations 2000. These require that the laboratory is registered with the Health and Safety Executive for the performance of GMO work, and that various other safeguards are put in place, before any GMOs enter the laboratory. Many other countries also have regulations/legal requirements covering work with GMOs.)

These are extremely important considerations as the relevant health and safety legislation may require (or recommend) features that need to be incorporated into the laboratory design from the start (e.g. specific air handling, the need for a changing lobby, accessibility of autoclaves), as well as constraining the specifications of the equipment required within the laboratory.

1.2.1.2 The scale of the work

• How many and of what size are the largest vessels that will be used within the facility?
• Will they require support vessels? (For example, a large fermenter would also require media preparation and storage vessels as well as smaller fermenters in which to grow up the cell inoculum.)
• For both of the above, will they be fixed or mobile? Is special handling equipment required? How will they be cleaned and/or sterilized?
• How are any spillages to be dealt with?

Thought needs to be given to the safe preparation, handling, inactivation and disposal o...