How to set-up a protocol for microcarriers

Background

Counting cells on microcarriers is a difficult challenge for everyone working in this field. Automated cell counters cannot provide a solution. Usually, microscopes or other time-consuming preparation methods are used to count cells on microcarriers. These methods are very complicated, time-consuming, non-robust and results significantly varies between different samples/users. 

We have created an exclusive application to solve this challenge and generate a robust method to count cells on Microcarriers using the NucleoCounter family.    

How To set-up a protocol for microcarriers

  • First you need to know that this will differ from customers to customers depending on the used methods/Culture and he wants to see.
  • The aim is to detach the cells from the microcarrier surfaces, thus both lysis buffers A100 and B is needed.
  • A general, step to step application notes for microcarriers can be found in here:

Using Cassettes:

https://marketing.chemometec.com/acton/attachment/21287/f-0167/1/-/-/-/-/994-0221%20v%201.2%20Tech%20Note%200221%20Count%20of%20Microcarrier%20Cultured%20Cells%20%28NC-200%29.pdf

Using A2 slides:

https://marketing.chemometec.com/acton/attachment/21287/f-016a/1/-/-/-/-/994-3815%20v%201.0%20Tech%20Note%20Count%20of%20Microcarrier%20Cultured%20Cells%20using%20A2-Slide%20%28NC-250%29.pdf

Set up a protocol:

In principal, you need to add solution A100 to the microcarriers sample, suspend and incubate for an indicated time, then adding solution B, suspend and let the microcarriers to sediment for an indicated time. Finally, counting using the protocol ‘’Count of Aggregated cells – A100 and B Assays’’

If viability detection is also desired, you will use the protocol ‘’Viability and Cell count – A100 and B Assays’’.

Important facts:

How to decide for how long shall I incubate the microcarriers with the solution A100?

It is different between each user. Usually it varies between 30s and 2min. The user shall make a comparison study on incubations for different times (recommended 4 different time points: 30s, 1min, 1min30s and 2min). At each time point a sample of the microcarriers shall be taken after incubation and observed under optical microscope. This observation will allow us to verify the best time for incubation for all microcarriers to be cell free.

Please notice that a longer incubation time with the solution A100 would cause the damage of the nuclear membrane and therefore no interaction with DAPI. Therefore, the timing should be kept exact for each time sample count to maintain a good reproducibility.

How to decide for how long shall I let the microcarriers to sediment after adding the solution B?

Once you have achieved to find the best time of incubation with solution A100, it is mandatory to add rapidly the solution B and let the microcarriers sediment in order to have a supernatant of cell nucleus only.

Since there is different type of microcarriers and it could differ in materials and Mass, the speed of sedimentation would vary correspondingly. But what is for sure, is all microcarriers are heavier of a nucleus and would sediment much faster. Also, the sample volume would for sure affect the sedimentation time 

In order to eliminate the sedimentation of the Nuclei also, you have to make sure that you are waiting for the right moment before taking out your supernatant.

Here you have to consider different time points to be tested (We recommend 6 different time points for a working volume of 1 ml: 30s, 1min, 1min30s, 2min, 3min, 4min)

After each sedimentation time point, you have to look at the bottom of your assay tube to see if a visible pellet is form, which is an indication for the sedimentation of the microcarriers. Additionally, you can separate the supernatant formed, resuspend it and look under optical microscope for the presence of microcarriers. In here also, exact timing is important to be considered for each sample counting to avoid strange variations. 

Shall I always aliquot the supernatant (containing the nuclei) in a new assay tube?

We highly advise that to be sure that you are not taking any risks, especially when you have to perform technical replicates from the same sample, where you will be able to easily suspend your supernatant in order to obtain a homogeneous solution all the times.  

Can temperature affect the lysis process?

Usually RT should be enough for the incubation with solution A100. We have encountered in some cases, slight variations after incubation under 37°C or physiological environment. Therefore, we cannot give you a 100% answer.  

In here, is up to you to test both variations on your experimental set up in order to set up the right protocol. You can simply add the temperature parameters while testing the different time points of incubation and check if it may add some effect. 

How to proceed to detect the viability?

In here you will be using two measurements either with cassettes or A2-slides. After selecting the appropriate protocol ‘’Viability and Cell count – A100 and B Assays’’ and pressing on run, you will automatically see notices windows that pop-ups on your NucleoView software. Follow these notices by inserting first your lysed solution, where the DAPI signal will be presenting the total cell count. Afterward, the software will ask to insert your non-lysed samples (original samples), where the DAPI signal will be presenting only the dead cells that are practically detached from the Microcarriers. The software then will automatically use these two generated values to calculate the percentage of viability.  

I have attached also a comprehensive study done by Hookipa biotech on counting the microcarriers using the NucleoCounter NC-3000.

Last updated byAnonymous