How to run a NC-200/NC-250/NC-3000 demo for microcarriers

Background

Microcarriers offer a convenient method for growing adherent cells in bioreactors, scaling up cell and virus production on industrial scale. Cells grown on microcarriers have traditionally been counted using a multistep process that involves trypsin digestion and trypan blue staining. This method is both time consuming (40 min) and inaccurate(aggregates, incomplete release). Using NucleoCounting the entire cell counting process is completed in less than 5 minutes, which significantly saves time when counting cells in microcarrier cultures.

In order to determine the cell concentration, a sample containing cells in suspension is diluted with Reagent A100 (lysis buffer) followed by stabilization with Reagent B and drawn into the Via1-Cassette™. The addition of Reagent A100 will release the nuclei into suspension, which are then stabilized with Reagent B.

How to run a NC-200 demo for microcarriers

To run a demo for microcarriers on NC-200, we would like to demonstrate:

  • CV: use triplicates at each measurement to calculate CV values. 
  • Linearity: dilution curve. Dilute microcarrier cell suspension in culture media to generate a series of dilution ratios. Check for correlation between expected and obtained values.

Materials needed:

  • Microcarrier cell suspension culture to be counted (2 samples)
  • Reagent A100 
  • Reagent B 
  • Two Via1-Cassettes™

To determine Viability and Cell Count for microcarrier cultures, the procedure to follow is found in the “Viability and Cell Count of Microcarrier Cultured Cells” (994-0221):

1. The first step is to determine the total cell concentration of the microcarrier cell sample. 

  1. Stir the microcarrier-cell culture vessel to obtain a homogenous suspension and add one volume of Reagent A100. For example, to 100 µl of cell suspension add 100 μl of Reagent A100. Mix by pipetting. 
  2. Add 1 volume of Reagent B to the mixture of cell suspension and Reagent A100e.g. to 200 µl of the mixture of cell suspension and Reagent A100 add 100 μl of Reagent B. Mix by pipetting. 
  3. To avoid clogging of the Via1-CassetteTM, leave the microcarrier cell sample diluted 1:1:1 with the Reagent A100 and the Reagent B in a rack for 1-2 min until the microcarriers have settled in the bottom of the tube.
  4. When the microcarriers have settled to the bottom of the tube, insert the Via1-Cassette™ halfway into the liquid and press the piston to collect a sample (Figure 1).  
  5. Select the “Viability and Cell Count – A100 and B Assay” and press RUN. Insert the loaded Via1-Cassette™ containing the sample diluted 1:1:1 with Reagent A100 and Reagent B into the NucleoCounter® NC-200™ and click “OK’.  

2. The second step is to determine the concentration of non-viable cells in the undiluted cell sample

  1. Mix the undiluted microcarrier cell suspension to obtain a homogenous suspension by pipetting and transfer at least 100 µl to a separate vial (without the Reagent A100 and Reagent B treatment).
  2. To avoid clogging of the Via1-Cassette, incubate the undiluted microcarrier cell sample in a rack for 1-2 min.
  3. When the microcarriers have settled to the bottom of the tube, insert the Via1-Casstte™ halfway into the liquid and press the piston to collect a sample (Figure 1).  
  4. When prompted by a message box, replace the first Via1-Cassette™ with the second Via1-Cassette™ loaded with the undiluted cell suspension and click “OK“. 

Figure 1. Correct loading of the Via1-CassetteTM

Notes:

For macroporous microcarriers, Reagent A100 incubation time on the bench needs to be evaluated in order to achieve complete release of nuclei from the microcarrier support. It is recommended to perform a time course experiment where the microcarrier-cell suspension is incubated in Reagent A100 for up to 10 min. The optimal incubation time should be incorporated into the microcarrier counting procedure.

To determine Total Cell Count without Viability, only the first step needs to be performed and “Count of Aggregated Cells – A100 and B Assay” has to be selected.

How to run a NC-250/ NC-3000 demo for microcarriers

To run a demo on NC-250/NC-3000 for microcarriers, we would like to demonstrate:

  • CV: use triplicates at each measurement to calculate CV values. 
  • Linearity: dilution curve. Dilute microcarrier cell suspension in culture media to generate a series of dilution ratios. Check for correlation between expected and obtained values.

Materials needed

  • Cells to be counted (2 samples)
  • NC-Slide A2™ 
  • Reagent A100 (Lysis buffer)
  • Reagent B (Stabilizing buffer) 
  • Solution 12 (500 µg/ml DAPI) 

Sample 1: Total cell count 

  1. Add 1 volume of Solution 12 into 99 volumes of Reagent A100 e.g., add 10 μl of Solution 12 to 990 μl Reagent A100. Note: do not store this mixture, but prepare a new each time the assay is performed. 
  2. The original cell suspension is mixed to obtain a homogenous suspension. Pipette a representative cell sample from the cell suspension into a microcentrifuge tube (e.g. 100 µl).
  3. Add 1 volume of the mixture of Solution 12 and Reagent A100 to the microcentrifuge tube with the cell sample e.g., if the volume of the cell sample is 100 μl then add 100 μl of the mixture of Solution 12 and Reagent A100. Mix by pipetting. 
  4. Add one volume of Reagent B to the mixture of cell suspension, Reagent A100 and Solution 12 e.g. to 200 µl of the mixture of cell suspension, Reagent A100 and Solution 12 add 100 μl of Reagent B. Mix by pipetting. 
  5. Load 30 µl of the cell sample for the total cell count into chamber 1 of the slide.  

Sample 2: Non viable cell count: 

  1. Mix the original cell suspension again to obtain a homogenous suspension. Pipette a representative cell sample from the cell suspension into a microcentrifuge tube (e.g. 199 μl). 
  2. Add 1 volume of Solution 12 to 199 volumes of cells sample in the microcentrifuge tube e.g., if the volume of the cell sample is 199 μl then add 1 μl of Solution 12. Mix by pipetting. 
  3. Load 30 µl of the sample for the non-viable cell count into chamber 2 of the slide. 
  4. Place the loaded slide on the tray of the NucleoCounter® NC-250™ and select “Viability and Cell Count – A100 and B Assay” and sample unit NC-Slide A2™ and press RUN.

Notes:

For macroporous microcarriers, Reagent A100 incubation time needs to be evaluated in order to achieve complete release of nuclei from the microcarrier support.

To determine Total Cell Count but not Viability, only the first step needs to be performed and “Count of Aggregated Cells – A100 and B Assay” has to be selected.

It is also possible to count cells grown on microcarriers with Via1cassette on the NC-3000 performing the same procedure as on the NC-200.

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