I am dealing with human epithelial cancer lines cultured in T-75 flasks. After trypsinisation, trypsin inactivation and centrifugation, the cell pellets collected were resuspended 30-40 times with fresh media before subculture/seeding. Nevertheless, clumps of cells can be observed along with single cells.
Kindly advice to avoid the cell clumps.
1) Any better angles for the tubes containing pellet and pipette during resuspension?
2) The resuspended cells are 20mL in volume. Which serological pipette would
Hello Thai Leong Lai
Cell clumps are formed due to the following reasons:
1. Overgrowth of cells in culture causes excessive build-up of cell debris and free DNA released from dead cells. The sticky nature of DNA causes cells and other debris to aggregate into large clumps.
2. Excessive treatment with proteolytic enzymes like trypsin during trypsinization may lead to cell death resulting in the release of DNA from dead cells causing cells to clump.
3. Avoid environmental stress to cells in culture as this can accelerate cell death which could again lead to cell-cell adhesion causing clumping.
4. During cell pelleting, centrifuge cells at an optimum speed. Avoid high speed centrifugation which can lead to cell death thereby resulting in cell clumping.
Subculture cells when they are 80% confluent. Do not wait till they are overconfluent. Expose cells to trypsin for a maximum of 5 minutes. Avoid environmental stress by replacing old culture medium with fresh medium every second or third day.
So, when you are handling cells in culture as far as possible follow the above mentioned points.
You should be able to resolve this problem. In case you are still unable to solve the problem, you may try using DNase I at concentrations from 20-100µg/ml to avoid formation of clumps. For each cell type, the working concentration must be determined individually.
If there are minor few clumps, they can be taken care of by pipetting the cell suspension gently up and down few times.
Best.
You should be breaking up clumps in a small volume (1-2mL of media) using a p1000 pipet prior to adding to your final volume of 20mL. It is extremely hard to break up clumps in a large volume with a serological pipet. If you still cannot get a uniform single cell suspension then you can run the cells through a cell strainer.
Make sure you are using optimum amount of trypsin so as to cover the whole cell layers and the edges. You can swirl the contents frequently during the process.
You can try passing the cells through a strainer after you resuspend them, same as they do for FACS or FlowCyt.
I agree with Kristin M. Adams. You need to add 1 or 2 ml media to the cell pellets. Mix well by pipetting with a 1 ml pipette before going for a large volume. I am also doing it in the same way.
Try resuspending cells in small volume of media and in case you need more then you can add the media later. This will help in proper distribution of trypsinized cells and they won't form clumps.
If the final volume is 20ml, dissolve the cell pellet in less than 10ml of fresh media in a 50ml falcon tube, use a 5ml serological pipette for resuspension at least 50times, before adding the remaining of fresh media.
Observe your trypsinization process, try different incubation timings with different trypsin volumes. For eg. 2ml-1:30mins or 1.5ml-2mins. Check the cells under microscope if they are detaching in clumps.
Hi Thai Leong Lai,
While resuspending the cell pellet, make sure you resuspend in a sufficient volume ( for 20ml final volume, 5 ml should be enough) and mix thoroughly. When using a pipette gun make sure you are gently mixing the cell and not rupturing them. In most cases this is enough for all applications, if the trypsination was carried out correctly ( 2.5ml for T75 flask and incubated in an incubator for an appropriate time depending on the cell line).
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