Part a

Intro to Cell Culture

This is Part A, Intro to Cell Culture, under the module topic, Cell Culture Techniques. This topic part has one section: Content Tutorial.

Content Tutorial

INTRODUCTION: Studying Cells – The Arizona Biology Project

The following web link will take you to the Arizona Biology Project’s Studying Cells Tutorial. The tutorial provides a basic overview about the study of cells that includes some background information reviews cell theory, cell sizes, and some basic information about different types of microscopes used to study cells.

• Studying Cells Tutorial (www.biology.arizona.edu, HTML Page)

Use the following link to learn more about working with cells

• Content Tutorial (www.research.umbc.edu, HTML Page)

Cell Culture Overview:

I Types of cells grown in culture II Work area and equipment III Preservation and storage IV Safety considerations

I. TYPES OF CELLS GROWN IN CULTURE

Tissue culture is often a generic term that refers to both organ culture and cell culture and the terms are often used interchangeably. Cell cultures are derived from either primary tissue explants or cell suspensions. Primary cell cultures typically will have a finite life span in culture whereas continuous cell lines are, by definition, abnormal and are often transformed cell lines.

II. WORK AREA AND EQUIPMENT

A. Laminar flow hoods. There are two types of laminar flow hoods, vertical and horizontal. The vertical hood, also known as a biology safety cabinet, is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment. Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures. Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion. NOTE: these are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work. The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light. Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10-20 minutes before being used. Wipe down all surfaces with ethanol before and after each use. Keep the hood as free of clutter as possible because this will interfere with the laminar flow air pattern.

B. CO2 Incubators. The cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times.

C. Microscopes. Inverted phase contrast microscopes are used for visualizing the cells. Microscopes should be kept covered and the lights turned down when not in use. Before using the microscope or whenever an objective is changed, check that the phase rings are aligned.

D. Preservation. Cells are stored in liquid nitrogen (see Section III- Preservation and storage).

E. Vessels. Anchorage dependent cells require a nontoxic, biologically inert, and optically transparent surface that will allow cells to attach and allow movement for growth. The most convenient vessels are specially-treated polystyrene plastic that are supplied sterile and are disposable. These include petri dishes, multi-well plates, microtiter plates, roller bottles, and screwcap flasks – T-25, T-75, T-150 (cm2 of surface area). Suspension cells are either shaken, stirred, or grown in vessels identical to those used for anchorage-dependent cells.

III. PRESERVATION AND STORAGE

Liquid N2 is used to preserve tissue culture cells, either in the liquid phase (-196oC) or in the vapor phase (-156oC). Freezing can be lethal to cells due to the effects of damage by ice crystals, alterations in the concentration of electrolytes, dehydration, and changes in pH. To minimize the effects of freezing, several precautions are taken. First, a cryoprotective agent which lowers the freezing point, such as glycerol or DMSO, is added. The freezing medium we typically use is 90% serum, 10% DMSO. In addition, it is best to use healthy cells that are growing in log phase and to replace the medium 24 hours before freezing. Also, the cells are slowly cooled from room temperature to -80oC to allow the water to move out of the cells before it freezes. The optimal rate of cooling is 1o- 3oC per minute. Some labs have fancy freezing chambers to regulate the freezing at the optimal rate by periodically pulsing in liquid nitrogen. We use a low tech device called a Mr. Frosty. The Mr. Frosty is filled with 200 ml of isopropanol at room temperature and the freezing vials containing the cells are placed in the container and the container is placed in the -80oC freezer. The effect of the isopropanol is to allow the tubes to come to the temperature of the freezer slowly, at about 1oC per minute. Once the container has reached -80oC (about 4 hours or, more conveniently, overnight) the vials are removed from the Mr. Frosty and immediately placed in the liquid nitrogen storage tank. Cells are stored at liquid nitrogen temperatures because the growth of ice crystals is retarded below -130oC. To maximize recovery of the cells when thawing, the cells are warmed very quickly by placing the tube directly from the liquid nitrogen container into a 37oC water bath with moderate shaking. As soon as the last ice crystal is melted, the cells are immediately diluted into prewarmed medium.

IV. SAFETY CONSIDERATIONS

Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should also be observed:

• pipetting: use pipette aids to prevent ingestion and keep aerosols down to a minimum •no eating, drinking, or smoking •wash hands after handling cultures and before leaving the lab •decontaminate work surfaces with disinfectant (before and after) •autoclave all waste •use biological safety cabinet (laminar flow hood) when working with hazardous organisms. The cabinet protects worker by preventing airborne cells and viruses released during experimental activity from escaping the cabinet; there is an air barrier at the front opening and exhaust air is filtered with a HEPA filter make sure cabinet is not overloaded and leave exhaust grills in the front and the back clear (helps to maintain a uniform airflow) •use aseptic technique •dispose of all liquid waste after each experiment and treat with bleach

References: R. Ian Freshney, Culture of Animal cells: A manual of basic techniques, Wiley-Liss, 1987.

Part b

Aseptic Technique

This is Part B, Aseptic Technique, under the module topic, Cell Culture Techniques. This topic part has two sections: ContentTutorials & Animations.

Content Tutorials

A. Purpose: To describe aseptic techniques used by Cell Culture specialists in handling products from and/or mammalian cells. B. Safety: Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material. Use gloves, goggles, mask, and protective clothing as necessary. C. Equipment: Laminar flow or biological safety hood as appropriate to the hazardous nature of the project. D. Materials: Wipes- lint-free, disinfectant or quaternary ammonium, Alcohol – 70% ethanol, Pipets – sterile and of appropriate size, Pipet-Aid or equivalent, Biohazard waste container. E. Procedure:

1. Carry out all culturing operation is a laminar flow hood.
2. Disinfect all surfaces prior to use with a disinfectant solution.
3. Swab down the working surface liberally with 70% ethanol.
4. Periodically spread a solution of 70% ethanol over the exterior of gloves to minimize contamination. Replace them if torn.
5. In case of any spill, spread a solution of 70% alcohol and swab immediately with non-linting wipes.
6. Discard gloves after use and do not wear them when entering any other lab area.
7. Bring into the work area only those items needed for a particular procedure.
8. Leave a wide clear space in the center of the hood (not just the front edge) to work on. Do not clutter the area to prevent blockage of proper air flow and to minimize turbulence.
9. Swab with 70% alcohol all glassware (medium bottles, beakers, etc.) before placing them inside the hood.
10. Arrange the work area to have easy access to all of it without having to reach over one item to get at another (especially over an open bottle or flask).
11. Use sterile wrapped pipets and discard them after use into a biohazard waste container.
12. Check that the wrapping of the sterile pipet is not broken or damaged.
13. Inspect the vessels to be used

T-flask – Must be free from visible contamination or breakage, or lack container identification. The plastic covering the flask must be intact. Bottles – Check for cracks, expiration dates. Spinner flasks – Check for cracks, expiration dates, and proper assembly.

14. Discard any biohazard or contaminated material immediately.
15. Never perform mouth pipetting. Pipettor must be used.
16. When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
17. Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.
18. Clean the work area when finished by wiping with 70% alcohol.

Additional Aseptic Technique Guidelines: UMBC Applied Molecular Biology, Julie B. Wolf

• Sterile Technique Methods (userpages.umbc.edu, HTML Page)

Animations

1. Video Cell Culture: Aseptic Technique (Reference, Sigma-Aldrich) Click on the following link to view a video depicting appropriate practices for maintaining aseptic technique when working with cell culture experiments. The video displays the equipment that is typically used in cell culture experiments and the website also contains important text located below the video image box highlighting the major points from the video.

• Cell Culture Video (www.sigmaaldrich.com, Multimedia Page)

2. Cell Culture Contamination: University of North Carolina Images Index of Typical Cell Culture Contaminants (Bacteria, Fungus, Mycoplasma, Yeast)

• Cell Culture Contamination Images (www.unclineberger.org, Multimedia Page)