Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)
Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization.
We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and FCCP to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions.
The validation of the method consisting in a JURKAT cell line experimental incorporation as internal control contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.
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Posted 18 Sep, 2020
On 06 Jan, 2021
On 04 Nov, 2020
On 01 Nov, 2020
Received 25 Oct, 2020
Received 18 Oct, 2020
Received 17 Oct, 2020
On 05 Oct, 2020
On 05 Oct, 2020
On 04 Oct, 2020
Invitations sent on 03 Oct, 2020
On 03 Oct, 2020
On 25 Sep, 2020
On 24 Sep, 2020
On 16 Sep, 2020
On 11 Sep, 2020
Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)
Posted 18 Sep, 2020
On 06 Jan, 2021
On 04 Nov, 2020
On 01 Nov, 2020
Received 25 Oct, 2020
Received 18 Oct, 2020
Received 17 Oct, 2020
On 05 Oct, 2020
On 05 Oct, 2020
On 04 Oct, 2020
Invitations sent on 03 Oct, 2020
On 03 Oct, 2020
On 25 Sep, 2020
On 24 Sep, 2020
On 16 Sep, 2020
On 11 Sep, 2020
Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization.
We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and FCCP to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions.
The validation of the method consisting in a JURKAT cell line experimental incorporation as internal control contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6