Basics of Cell Culture

Introduction

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial controlled environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation. The other way is to drive them from a cell line or cell strain that has previously been established.

Primary culturing:

Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they reach the confluence stage. At this stage, the cells have to be subcultured by transferring them to a new vessel with a fresh growth medium to provide more room for continued growth.

Cell Line:

The primary culture is known as a cell line or subclone after the first subculture. Cell lines derived from primary cultures have a finite life span and as they are passaged, cells with the highest growth capacity predominate, which ultimately results in a degree of genotypic and phenotypic uniformity in the population.

Cell Strains:

If the subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.

Finite cell line:

Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite.

Infinite cell line:

 Some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.

Culture Conditions:

 Culture conditions may vary widely for each cell type, but the artificial environment in which the cells are cultured consists of a suitable vessel containing a  medium. that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (O2, CO2), and regulates the physicochemical environment (pH, osmotic pressure, temperature). Most cells are anchorage dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).

Cryopreservation

 If a surplus of cells is available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO with glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed.

Cell Morphology:

 Morphology of the cells in culture (i.e., their shape and appearance) is essential for successful cell culture experiments. In addition to confirming the healthy status of your cells, inspecting the cells through a microscope each time they are handled will allow us to detect any signs of contamination early on and to contain it before it spreads to other cultures around the laboratory. Signs of deterioration of cells include granularity around the nucleus, detachment of the cells from the substrate, and cytoplasmic vacuolation. Signs of deterioration may be caused by a variety of reasons, including contamination of the culture, senescence of the cell line, or the presence of toxic substances in the medium, or they may simply imply that the culture needs a medium change. Allowing the deterioration to progress too far will make it irreversible.

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On the basis of shape cells in culture are divided into three basic categories:

• Fibroblastic cells: are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.

 • Epithelial-like cells: are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.

 • Lymphoblast-like cells: are spherical in shape and usually grown in suspension without attaching to a surface.

Cell culture lab equipements:

The specific requirements of a cell culture laboratory depend mainly on the type of research conducted in the laboratory. For example, the need for mammalian cell culture laboratory specializing in cancer research is quite different from that of an insect cell culture laboratory that focuses on protein expression. However, all cell culture laboratories have the common requirement of being free from pathogenic microorganisms (i.e., asepsis), and almost the same basic equipments are required that is essential for culturing cells.

The Equipments required in a cell culture lab

Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)	Incubator (humid CO2 incubator recommended)	Water bath	Centrifuge
Refrigerator and freezer (–20°C)	Cell counter	Inverted microscope	Liquid nitrogen (N2) freezer
Sterilizer (i.e., autoclave)	Aspiration pump	pH meter	Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-well plates)
Pipettes	Syringes and needles	Waste containers	Media, sera, and reagents

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 I, II, and III, have been developed to meet varying research and clinical needs.

 Classes of Cell Culture Hoods:

• 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 culture protection from contamination. They are similar in design and airflow characteristics to chemical fume hoods.
• Class II cell culture hoods are designed for work involving BSL-1, 2, and 3 materials, and they also provide an aseptic environment necessary for cell culture experiments. A Class II biosafety cabinet should be used for handling potentially hazardous materials (e.g., primate-derived cultures, virally infected cultures, carcinogenic or toxic reagents).
• Class III biosafety cabinets are gas-tight, and they provide the highest attainable level of protection to personnel and the environment. A Class III biosafety cabinet is required for work involving known human pathogens and other BSL-4 materials.

Air-Flow Characteristics of Cell Culture Hoods Cell culture hoods protect the working environment from dust and other airborne contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. The flow can be horizontal, blowing parallel to the work surface, or it can be vertical, blowing from the top of the cabinet onto the work surface. Depending on its design, a horizontal flow hood provides protection to the culture (if the air flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet by the negative air pressure inside). Vertical flow hoods, on the other hand, provide significant protection to the user and the cell culture.

Clean Benches:

 Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety cabinets; these pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the user, and they may expose the user to potentially hazardous materials. These devices only provide product protection. Clean benches can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices, and they should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials.

Cell Culture Hood Layout:

 A cell culture hood should be large enough to be used by one person at a time, be easily cleanable inside and outside, have adequate lighting, and be comfortable to use without requiring awkward positions. Keep the workspace in the cell culture hood clean and uncluttered, and keep everything in direct line of sight. Disinfect each item placed in the cell culture hood by spraying it with 70% ethanol and wiping it clean.

The arrangement of items within the cell culture hood usually adheres to the following right-handed convention, which can be modified to include additional items used in specific applications.

•  A wide, clear workspace in the center with your cell culture vessels.
• Pipettor in the front right, where it can be reached easily.
• Reagents and media in the rear right to allow easy pipetting.
• Tube rack in the rear middle holding additional reagents.
• A small container in the rear left holds liquid waste

Incubator:

 The purpose of the incubator is to provide the appropriate environment for cell growth. The incubator should be large enough for your laboratory needs, have forced air circulation, and should have temperature control to within ±0.2°C. Stainless steel incubators allow easy cleaning and provide corrosion protection, especially if humid air is required for incubation. Although the requirement for aseptic conditions in a cell culture incubator is not as stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to avoid contamination of cell cultures.

Types of Incubators:

 There are two basic types of incubators needed in cell culture laboratories. These include:

 Dry incubators:

These are more economical, but require the cell cultures to be incubated in sealed flasks to prevent evaporation. Placing a water dish in a dry incubator can provide some humidity, but they do not allow precise control of atmospheric conditions in the incubator.

 Humid CO2:

These incubators are more expensive, but allow superior control of culture conditions. They can be used to incubate cells cultured in Petri dishes or multi-well plates, which require a controlled atmosphere of high humidity and increased CO2 tension.

Storage:

 A cell culture laboratory should have storage areas for liquids such as media and reagents, for chemicals such as drugs and antibiotics, for consumables such as disposable pipettes, culture vessels, and gloves, for glassware such as media bottles and glass pipettes, for specialized equipment, and for tissues and cells. Glassware, plastics, and specialized equipment can be stored at ambient temperature on shelves and in drawers; however, it is important to store all media, reagents, and chemicals according to the instructions on the label. Some media, reagents, and chemicals are sensitive to light; while their normal laboratory use under lighted conditions is tolerated, they should be stored in the dark or wrapped in aluminum foil when not in use. 

Refrigerators:

 For small cell culture laboratories, a domestic refrigerator (preferably one without an autodefrost freezer is an adequate and inexpensive piece of equipment for storing reagents and media at 2–8°C. For larger laboratories, a cold room restricted to cell culture is more appropriate. We have to make sure that the refrigerator or the cold room is cleaned regularly to avoid contamination.

Freezers:

 Most cell culture reagents can be stored at –5°C to –20°C; therefore an ultradeep freezer (i.e., a –80°C freezer) is optional for storing most reagents. A domestic freezer is a cheaper alternative to a laboratory freezer. While most reagents can withstand temperature oscillations in an autodefrost (i.e., self-thawing) freezer, some reagents such as antibiotics and enzymes should be stored in a freezer that does not autodefrost.

Cryogenic Storage:

 Cell lines in continuous culture are likely to suffer from genetic instability as their passage number increases; therefore, it is essential to prepare working stocks of the cells and preserve them in cryogenic. Do not store cells in –20°C or –80°C freezers, because their viability quickly decreases when they are stored at these temperatures. There are two main types of liquid-nitrogen storage systems, vapor phase and liquid phase, which come as wide-necked or narrow-necked storage containers. Vapor phase systems minimize the risk of explosion with cryo storage tubes, and are required for storing biohazardous materials, while the liquid phase systems usually have longer static holding times, and are therefore more economical. Narrow-necked containers have a slower nitrogen evaporation rate and are more economical, but wide-necked containers allow easier access and have a larger storage capacity.

Applications of cell culture:

• It is a major tool in molecular and cellular biology.
• It helps to study normal cell homeostasis, cell biochemistry, metabolism, mutagenesis, diseases, and compound effects. It also helps in drug screening.
• The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.