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Cell Culture Techniques

In cell culture techniques, cells or tissues are removed from the organism (animal or plant) and introduced into an artificial environment with ideal conditions for growth. This procedure helps scientists and researchers to study and understand more about the cells.

It ultimately requires the distribution of cells in an artificial (in vitro) environment consisting of the nutrients, ideal temperature, gases, PH and humidity essential to expand and proliferate the cells. There are two major types of cell culture media:

• In vivo – When the study involves living biological entities within the organism
• In vitro – When the study involves using biological entities (cells, tissues, etc) that were isolated from their natural environment

What is Cell Freezing?

In continuous culture, cell lines are bound to suffer undesirable results such as genetic drift, senescence, and microbial contamination, and equipment failure can occur even in the best-run laboratories.

One of the benefits of operating in vitro is the ability to freeze and retain cells in liquid nitrogen with limited loss of viability. If cells are lost due to infection, carelessness, equipment failure, or a natural disaster, having a frozen bank of cells provides a backup.

In large batches, certain types of primary cultures can be prepared, a large sample of vials frozen, and the cells sequentially thawed and examined as secondary cultures, so many experiments can be carried out from the same preparation on early passage cells.

Alternatively, when one attempts to create a cell line, every three to five passages should have a few vials frozen, to provide a permanent record of any changes that may occur with a passage number. A valuable resource is an existing cell line, and its replacement is costly and time-consuming. They must then be frozen down for long-term storage and preserved. An appropriately conserved frozen cell stock is a central part of cell culture.

Expansion and freezing of an early passage bank are the only methods that enable identical cells to be used in several different laboratories on typical human cells that have a short life span in vitro. Whenever a new cell line property is fully characterized or a cell line is recloned, a number of vials should be frozen so that if the lines being carried are modified, they can return to these cells.  

In industrial production using mammalian cell lines, enormous cell banks are prepared and thoroughly characterized as to the absence of adventitious agents and the cell properties strictly limited and carefully defined period to avoid any alteration in the cells during culture. 

Cells are frequently easier to ship as frozen vials rather than in flasks. 

Cell Freezing Technique

At BioConcept Ltd., they freeze their cell lines immediately after packaging, and cells are stored in cryotubes designated “Seed Stock”.

Cell freezing technique is used to preserve seed stocks of any given culture, to protect against “genetic wandering” and contamination, and to store a culture that is not in daily use for a long period of time.

BioConcept Ltd. recommends that a proportion of the culture material be stored and frozen as early as possible after collecting a fresh stock of cells from primary sources. This should preferably be achieved after receiving the cell culture within 1 or 2 passages.

A “seed” stock of culture material may be withdrawn at intervals, thereby ensuring a constant, identical source of cells for many years to come. From the seed stock, further aliquots are taken, frozen and designated as “working stock.” This “working stock” has enough ampoules for approximately one year. When the “working stock” has been depleted, another ampoule of seed stock is thawed and grown up to provide a new supply of working stock.

Unrestricted supplies of genetically similar material can be assured by using this cell freezing protocol. BioConcept Ltd. does not recommend handling cells for more than 10 passages or 10 weeks in the laboratory. This ensures that changes will not occur in the cell line. It is essential that fresh back-up stocks always be available in deep-freeze.

When freezing cells, the following recommendations of cell freezing technique ensure high cell viability:

1. Check that cells are rapidly growing and in a phase of exponential growth before freezing. If working with finite, fibroblast cultures, they should NOT have been “confluent” for more than 24 hours. If dealing with a continuous cell line, harvest it at 70–80% confluency. Suspension cells should be spun and harvested while in exponential growth.
2. Trypsinise the monolayer as quickly as possible, using cold trypsin to minimise carryover. Use only minimal amounts of trypsin, ensuring that most are removed from the monolayer. This may be achieved by pouring off the trypsin or by centrifuging the cells and resuspending them in fresh medium.
3. Freeze the cells at a concentration between 2 and 5×106 cells/ml. For finite cultures, it is recommended that the entire contents of a flask be harvested, spun and the cell pellet resuspended in enough medium to produce one ampoule of material (i.e. enough to generate one flask worth of cells at passage number 15). If two ampoules are produced from one flask, then this must be accounted for in the passage number, which in this case, a 1:2 split has been accomplished, so the passage number would be 16.
4. For established cultures, the yield is often large enough for several ampoules to be produced from one parent flask. In this case, the passage number should be increased by 1 to indicate that a passage or the equivalent of a subcultivation has occurred.
5. Keep the monolayer suspension COOL to minimize damage from the cryoprotective reagent. Since suspension cells do not respond well to low temperatures, they should NOT be cooled. Place the ampoule in a controlled rate freezer that is set to cool at a rate of 1°C per minute. Allow the cells to freeze for approximately four hours before transferring them to a permanent position in the nitrogen freezer.
6. Frozen cells may be kept for short periods (4–10 weeks) at −20°C, but long-term storage should always be in the vapour phase of liquid nitrogen or an equivalent temperature.

Frozen Culture Recovery

1. Thaw the frozen ampoules by gently swirling the vial in a 37°C water bath until there is just a small bit of ice left in the vial.
2. Before proceeding with the thawing procedure, transfer the vial into a laminar flow hood, wipe the outside of the vial with 70 % ethanol or isopropanol to decontaminate it.
3. Remove the cells with a narrow pipette or syringe and transfer them dropwise over 1–2 minutes to a centrifuge tube or a culture flask filled up with pre-warmed complete medium appropriate for your cell line.
4. A 1:10 dilution should be made.
5. Centrifuge the cell suspension at approximately 200×g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
6. Gently resuspend the cell pellet in fresh growth medium and transfer them into the appropriate cell culture vessel.
7. If the cryoprotective reagent is NOT removed by centrifugation it is recommended to feed the cell culture after 24h incubation with the growth medium.

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