Human Pluripotent Stem Cells

A Practical Guide

Uma Lakshmipathy, Chad C. MacArthur, Mahalakshmi Sridharan, Rene H. Quintanilla

• Publisher: Wiley-Blackwell
• Year: 2017
• ISBN:
  9781119394365
• Edition: 1
• Topic: Ciencias biológicas
• Subtopic: Biología celular

Comprehensive coverage of the entire induced pluripotent stem cell basic work flow

Pluripotent stem cells (PSC) can divide indefinitely, self-renew, and can differentiate to functionally reconstitute almost any cell in the normal developmental pathway, given the right conditions. This comprehensive book, which was developed from a training course, covers all of the PSCs (embryonic, embryonic germ, and embryonic carcinoma) and their functions. It demonstrates the feeder-dependent and feeder-free culture of hESC and hiPSC, which will be referred to in all protocols as PSCs. It also addresses the methods commonly used to determine pluripotency, as defined by self-renewal marker expression and differentiation potential.

Human Pluripotent Stem Cells: A Practical Guide offers in-depth chapter coverage of introduction to stem cell, PSC culture, reprogramming, differentiation, PSC characterization, and more. It also includes four appendixes containing information on reagents, medias, and solutions; common antibodies; consumable and equipment; and logs and forms.

• Includes helpful tips and tricks that are normally omitted from regular research papers
• Features useful images to support the technical aspects and results visually as well as diagrammatic illustrations
• Presents specific sections (ie: reprogramming, differentiation) in a concise and easily digestible manner
• Written by experts with extensive experience in stem cell technologies

Human Pluripotent Stem Cells: A Practical Guide is an ideal text for stem cell researchers, including principal investigators, and others in university and industry settings, and for new graduate students in PSC labs.

1
Introduction

Successful execution of any cell‐based project relies on a setting up a robust cell culture laboratory. Guidelines under the Guidance on Good Cell Culture Practice provides an overview of the critical parameters in establishing facilities and training personnel [1]. This is even more important for a stem cell laboratory where primary cells from donor tissue or their derivatives are cultured for extended periods of time [2].

1.1 Biosafety

In addition to the safety risks common to most workplaces, such as electrical and fire hazards, a cell culture laboratory has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive or mutagenic solvents and reagents. The most common of these hazards are accidental inoculations with syringe needles or other contaminated sharps, spills and splashes onto skin and mucous membranes, ingestion through mouth pipetting, animal bites and scratches, and inhalation exposures to infectious aerosols.

The fundamental objective of any biosafety program is to reduce or eliminate exposure of laboratory workers and the outside environment to potentially harmful biological agents. The most important element of safety in a cell culture laboratory is strict adherence to standard microbiological practices and techniques.

1.2 Biosafety Levels

The regulations and recommendations for biosafety in the United States are contained in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH), and published by the US Department of Health and Human Services. The document defines four ascending levels of containment, referred to as biosafety levels 1 through 4, and describes the microbiological practices, safety equipment, and facility safeguards for the corresponding level of risk associated with handling a particular agent.

• Biosafety Level 1 (BSL‐1): the basic level of protection common to most research and clinical laboratories. Appropriate for agents that are not known to consistently cause disease in normal, healthy human adults (examples: Bacillus subtilis, E. coli).
• Biosafety Level 2 (BSL‐2): appropriate for moderate‐risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL‐2, and all stem cell labs have this as a requirement.
• Biosafety Level 3 (BSL‐3): BSL‐3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for agents that may cause serious and potentially lethal infections.
• Biosafety Level 4 (BSL‐4): BSL‐4 is appropriate for exotic agents that pose a high individual risk of life‐threatening disease by infectious aerosols and for which no treatment is available. These agents are restricted to high‐containment laboratories.

For more information about the biosafety level guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th edition, which is available for downloading at www.cdc.gov/biosafety/.

1.3 Aseptic Technique

Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacteria, fungi, and viruses. Non‐sterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and dirty work surfaces are all sources of biological contamination.

Aseptic technique, designed to provide a barrier between the microorganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

1.3.1 Maintaining a Sterile Work Area

The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood.

• The cell culture hood should be properly set up, and located in an area that is restricted to cell culture, is free from drafts from doors, windows, and other equipment, and with no through traffic.
• The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.
• Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.
• For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.
• Using a Bunsen burner for flaming is not necessary or recommended in a cell culture hood.
• Leave the cell culture hood running at all times, turning it off only when it will not be used for extended periods of time.
• Practice good personal hygiene. Wash your hands before and after working with cell cultures. In addition to protecting you from hazardous materials, wearing personal protective equipment also reduces the probability of contamination from shed skin as well as dirt and dust from your clothes.

1.3.2 Aseptic Work Area

The major requirement of a cell culture laboratory is to maintain an aseptic work area that is restricted to cell culture work. Although a separate tissue culture room is preferred, a designated cell culture area within a larger laboratory can be used for sterile handling, incubation, and storage of cell cultures, reagents, and media. The simplest and most economical way to provide aseptic conditions is to use a cell culture hood (i.e., biosafety cabinet).

1.3.3 Cell Culture Hood

The cell culture hood provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures. Three kinds of cell culture hoods, designated as Class II, III, and I, have been developed to meet varying research and clinical needs.

• Class I cell culture hoods offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques, but they do not provide cultures with protection from cont...