May 25, 2022
Shahid Sadoughi University of Medical Siences
Specialized Cell Culture Laboratory

Specialized Laboratory of Cell Culture

Type of activity: research, educational

Laboratory manager: Dr. Majid Pourentezari

 

Main facilities and equipment in cell culture

Some special equipment and techniques are needed to maintain cell cultures. In fact, having more facilities means doing the process of cell culture as well as possible. In the cell culture section, we have tried to provide a suitable environment and equip it with the devices and equipment needed for cell culture to provide the required space for students interested in cell culture. Below are some of the tools and devices in this section, along with a brief overview of their application in cell culture.

Sterile Work Environment

A separate room should be provided as far as possible for cell culture work. This room should be free of any crowds and, if possible, have a filtered air conditioning in the workplace. Primary animal tissue and microorganisms should not be cultured near a cell culture laboratory, and a separate, specialized laboratory for sterile cell culture should be designed. Cell culture laboratory gowns should be worn at the laboratory entrance and clothing removed from this environment should not be re-entered into the laboratory. In case of severe sterility, a laminar hood is recommended to better maintain sterile conditions. Vertical hoods should be used if a hazardous chemical is used. However, for primary cultures and when no laminar hood or sterile chamber is available, a sterile space should be considered for the work. If the sterilization methods are properly observed and the cleanliness and order of the environment is maintained, the sterile work environment can be somewhat comfortable.

All work surfaces, tables and shelves and laminar hoods should be properly cleaned and sterilized regularly with 70% ethanol or a replacement cleaner. If there is no laminar hood or air filters in the workplace, the culturing work can be done on a clean table with the help of a flame to sterilize the work surface and the surrounding air.

Incubation equipment

In addition to laminar air filters and hoods and easy-to-clean desks, the cell culture laboratory will need an incubator to keep the cells at 30 to 40 degrees Celsius. The incubation temperature depends on the type of cells cultured. Insect cells grow best at about 30 degrees Celsius, while mammalian cells need at about 37 degrees Celsius. It is necessary to use incubators that use a main source or gas cylinder to supply CO2, because the air inside the incubator must always have between 2 and 5% CO2. In general, many cell lines are able to maintain their viability in an atmosphere containing 95% air and 5% CO2 with 99% relative humidity. The CO2 concentration should be in equilibrium with the sodium carbonate of the culture medium. Different culture media have different buffering capacities. If a CO2-equipped incubator is not available, the cultures can be temporarily stored in an impermeable flask. In so doing, the flasks must be well sealed with paraffin tape after CO2 spraying.

It is also possible to use different culture media that do not require a CO2-containing atmosphere, in which case a CO2-equipped incubator is not required. For example, hepatocytes are stored in their primary culture in the Leboviztz L-15 culture medium. It does not require CO2, but in this case, the culture flasks should not be sealed and impermeable, because hepatocytes require high amounts of O2 and over time the amount of oxygen in the medium without impermeable gas decreases. Most cell lines are stored at 36.5°C, although some cultures, including skin cultures, may require lower temperatures. The cultured cells generally survive at lower temperatures, but their survival temperature is rarely more than 2 ° C above normal, so the incubator must be adjusted to increase the temperature to approximately 38.5°C. To prevent cell death, the incubators are designed to regulate a uniform temperature; in other words, the temperature must eventually change by ±0.5 ° C at any set temperature. Therefore, incubators equipped with air circulation system can help maintain the uniformity of air in the incubator.

-20 Refrigerators & Freezers

Both of these devices are very important for storing liquid culture medium at 4°C and for enzymes (e.g. trypsin) and some other culture medium contents (such as glutamine and serum) at -20°C. Refrigerators will be needed to maintain culture media, buffers, and freezers to store serum prior to segregation, nutrients, and antibodies. These natural substances need -20 degrees Celsius for storage, but cells need liquid nitrogen or -70 degrees Celsius for storage.

Microscope

Because the culture is done in flasks or plates, a reverse light microscope will be required. Detection of morphological changes in any culture is crucial as this can be the first sign of spoilage in a culture. A very simple light microscope with x100 magnification will suffice for the daily work of cell counting, although better quality microscopes are needed for chromosomal analysis or automated radiographic work. Reverse microscope is also used to examine the flasks and plates of multi-welled plates from the bottom of each container. Both types of microscopes must be equipped with x10 and x20 lenses, and x40 and x100 lenses must be used for conventional microscopes. Additional equipment such as cameras, CCD video cameras, adapters and other accessories and UV facilities are also needed for other purposes.

Cultivation Containers

There are many types of disposable plastic containers for culture, but the most common are polystyrene containers. Except that all disposable plastic containers for cell culture should provide sufficient cell growth, but it is essential to ensure that the culture can grow in a new container as well. Experiments in this direction include growth curves and the time to reach a uniform cell layer.

The cells can be stored in petri dishes or flasks that are equipped with a number of features, including the fact that the flasks can be carbonated with CO2 and then tightly closed (impermeable), so that the need for using the CO2 incubator is removed. This is especially useful when the incubator is not working properly. Cell culture dishes are always selected according to the method of work. Sometimes, it may be necessary to replace a used culture medium with another culture medium. The choice of container depends on several factors: 1. Is the culture in a suspension or is it a single layer? 2. Cell function: does culture require CO2 or not? 3. Cost can also be a limiting factor. The function of the cell is proportional to the available surface space. Ensuring the growth of a uniform cell monolayer is very important in many cases, especially in multi-welled (96, 48 or 24) containers.

Washing and Sterilizing Equipment

The availability of a wide range of plastic cell culture containers has significantly reduced the need for washing. However, glassware such as pipettes should be soaked in the rinsing solution and then rinsed thoroughly by soaking in distilled water. Then, the process of drying and sterilizing them is done by putting some cotton in the inlet of the pipettes and completely enclosing them with autoclave paper and autoclaving them at 121°C for 20 minutes. Also, glass containers such as pipettes, conical flasks, Erlen flasks and measured cylinders (with an aluminum coating) are sterilized in an oven at 160°C for 1 hour. Sterilization indicators such as sterilizing adhesives (autoclave adhesives) are essential for any type of sterilized device to ensure effective operation of the above devices. Use autoclave bags for open and unpacked items. Aluminum foils are also a good case for packing these devices for autoclave.

Liquid Nitrogen/ -70 Freezer

For both cell lines that replicate frequently and lines that eventually die, culture samples must always be frozen for storage. This is done to prevent cell mutations and to protect the cell line against contamination and other adverse events.

The process of freezing cells is common to all cell types. Cells should be frozen in the ascending phase of growth with a suitable preservative, usually dimethyl sulfoxide (DMSO). The cells are usually first placed at -20 degrees for a few hours and then immersed in liquid nitrogen at -196 degrees (in small closed vials) or stored in the gas phase at the top of the liquid. Cases of cell destruction and death have been observed at -70 degrees, so the priority is to use liquid nitrogen. The vials can be frozen in a polystyrene box with 1-inch walls.

Filter Sterilization

Environments that do not have autoclave capability should be sterilized by passing through a membrane filter with a pore diameter of 0.22. These filters are designed in different sizes to filter a wide range of materials with different volumes. These filters can be purchased as disposable or as autoclaved filters with a suitable holder. Culture media, enzymes, hormones, cofactors, and bicarbonate buffers are examples of these non-autoclaved materials.

 

Cell Counting Facilities

It is not possible to track cell growth ocularly, but more accurate cell counts are required for most laboratory purposes. The most commonly used device for cell counting is the neobar homocytometer slide, which is primarily designed to count blood cells. This thick slide consists of a series of vertical and horizontal lines in the form of a grid, which counts the average number of cells in 4 cells, which themselves are composed of 16 smaller cells. After pipetting, the cell suspension is loaded on this slide to completely separate the cells adhering to each other. Proper piping of the cell suspension before loading is very important, in order to be more precise and to break the adherent cell masses, which makes counting difficult. The number of cells counted in these wells at 104 indicates the number of cells per cubic millimeter of suspension.

Date:
2021/08/24
Views:
86
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تاریخ بروزرسانی: 1401/03/03

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