Hi, everyone!
Could anyone share their experience with cryostorage?
I would like to know, how many vials of which passage do you usually freeze to maintain a cell line in culture? Both for immortalised and not immortalised cell lines. I usually freeze 1 million of cells per vial.
Thank you all in advance!
Hello Yuliya Dzekhtsiarova
Once a cell line has been developed or acquired and validated (i.e., shown to be authentic and uncontaminated), the first step to ensuring a reliable and reproducible supply of cells is the cryopreservation of these cells.
Healthy, actively growing cells should be used for cryopreservation. I always try to freeze large numbers of cells that have not been passaged in culture too many times. Therefore, cryopreservation of cells of the first few passages is important. For immortalized cells, which will proliferate indefinitely, you may start freezing cells from passage 2 onwards and not greater than passage 10.
On the other hand, finite cell lines at the first subculture have a limited lifespan, and after a certain number of cell divisions the cells will senesce. So, for finite cell lines, freezing cells at early passages is essential. You may freeze down about 20 vials consisting of 1.5ml cryovials, each containing 1–5 × 10^6 cells/ml. This will ensure a ready supply for many years. The number of vials of each or alternate passages to be frozen will also depend upon the space availability in the liquid nitrogen storage tank.
I usually passage cells 1–2 days prior to freezing to ensure that they are in a logarithmic or exponential phase of growth at the time of freezing. It is also recommended changing the growth medium 24 hours prior to freezing if the cells have not been passaged at that time. Also, you should use controlled-rate freezing, a method that involves a gradual cooling rate of -1°C/minute, before long-term storage as it will help maximize cell viability and integrity.
Best.
Hi Yuliya Dzekhtsiarova ,
To maintain a cell line, the number of vials required depends on several factors, including the growth characteristics of the cell line, the desired passage interval, and the purpose of the maintenance. Typically, researchers aim to maintain an adequate supply of viable cells while minimizing the risk of genetic changes or contamination.
Here's a general guideline for maintaining a cell line:
1. Passage Interval: Determine the passage interval, which is the time or number of cell divisions between each passage. This interval varies depending on the cell type and growth rate.
2. Cell Count: Calculate the approximate number of cells needed for each passage. This calculation involves considering the growth rate, the desired confluency at the time of passage, and any additional experiments or downstream applications requiring cells.
3. Vial Size: Determine the size of the vials or containers used for storing cells. The vial size can vary depending on the laboratory's protocols and the specific cell line. Common sizes range from 1.5 mL cryovials to larger containers.
4. Passage Volume: Decide on the volume of cells to transfer during each passage. This volume should provide enough cells for further growth without overcrowding or exhausting the nutrients in the culture medium.
Based on these factors, you can estimate the number of vials needed to maintain a cell line. Here's an example calculation:
Let's assume you have a cell line with a passage interval of seven days, and you require 1 × 10^6 cells for each passage. If you use 1.5 mL cryovials for storage, and your cells are grown to confluency in a T75 flask (75 cm^2), you can follow these steps:
1. Determine the number of passages required within a given timeframe. For example, if you want to maintain the cell line for three months (approximately 90 days) and the passage interval is seven days, you will need approximately 90/7 = 13 passages.
2. Calculate the total number of cells needed: 1 × 10^6 cells/passage × 13 passages = 13 × 10^6 cells.
3. Estimate the number of vials needed: Assuming each cryovial can hold approximately 1 × 10^6 cells, you would require approximately 13 vials (rounded up to the nearest whole number) to store the cells.
It's important to note that these calculations serve as a general guideline, and the exact requirements may vary based on the specific cell line, laboratory protocols, and research objectives. Hope this helps!
Malcolm gave a good summary. In the last paragraph of his suggestion he mentions "...a method that involves a gradual cooling rate of -1°C/minute, before long-term storage as it will help maximize cell viability and integrity." A popular and robust way of doing this is to use "Mr. Frosty" (yes, it's a thing !). This will help your cells get acclimated to the temperature decline before liquid nitrogen transfer.
https://www.thermofisher.com/order/catalog/product/5100-0001
Thank you Can Kiessling for your comments as well as for the information on Mr. Frosty.
As others mentioned it depends on your aim for freezing but it is so important to freeze fresh cells. Its number can be from 100.000 to 1.000.000
The number of vials and passages required to maintain a cell line can vary depending on the specific cell line, experimental requirements, and laboratory protocols.
For Immortalized Cell Lines:
For Non-immortalized Cell Lines:
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