Assessing cytokine supplementation to improve lymphocyte viability after cryopreservation.
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Biology
Other
Neuroscience
Advisor Information
Paul W. Denton (PI)
Location
MBSC Ballroom Poster # 1104 - U
Presentation Type
Poster
Start Date
24-3-2023 10:30 AM
End Date
24-3-2023 11:45 AM
Abstract
Title: Assessing cytokine supplementation to improve lymphocyte viability after cryopreservation.
Bella Circo1, Anna R. Mahr1, Alexander K. Regan1, and Paul W. Denton, PhD1
Background: A primary goal of the Denton Immunobiology Lab is improving the killing efficacy of natural killer (NK) cells. The source for our NK cells is recently collected human PBMCs
available, we desire to use cryopreserved PBMCs. However, NK cell viability within recently thawed PMBCs has been poor. To address this, we hypothesized that exogenous cytokine stimulation could allow for testing with cryopreserved cells so long as cytokine levels can be identified that lead to improved viability without altering activation state during post-thawing incubations.
Methods: Purified human IL-2 and IL-15 were obtained. We established an overlapping matrix strategy whereby recently thawed PBMC exposure to each cytokine was independently titrated at the picomolar scale. Fresh PBMC were obtained. The fresh cells were divided into a fresh control arm and into a cryopreserved arm. Fresh samples were incubated overnight in complete RPMI media (RPMI + 10% fetal bovine serum+ 1% penicillin/streptomycin). Following the overnight incubation, cell viability was determined using trypan blue exclusion. For the cryopreserved samples, multiple aliquots were frozen back identically using standard protocols. This allowed for multiple cytokine matrices to be tested with overnight incubations using recently thawed PBMCs while simultaneously controlling for inter-human variability.
Results: Current results indicate that cytokine supplementation treatment at certain titrations yields higher variability than control PBMCs. Specifically, our data suggest that IL-2 supplementation alone is leading to higher viability in comparison to IL-15 supplementation alone. Furthermore, our data are showing that the combination dosages show maximal viability when IL-2 concentrations are increased while IL-15 concentrations decreased. Data-to-date will be shown.
Conclusions: In this study, we are striving to determine whether cytokine supplementation at picomolar concentrations can lead to cellular hemostasis post-cryopreservation of PBMCs. Future work will finalize our determination of an optimal cytokine supplementation strategy and we will also explore additional supplementations (e.g., DNAase) in the media to further improve viability. Next steps will be to verify that the supplementation strategy is not leading to exogenous activation of PBMCs during this “resting” overnight culture. Once conclusions for this project are finalized, we will produce a standardized lab protocol for future experiments that involve culturing cryopreserved PBMCs.
Scheduling
9:15-10:30 a.m., 10:45 a.m.-Noon
Assessing cytokine supplementation to improve lymphocyte viability after cryopreservation.
MBSC Ballroom Poster # 1104 - U
Title: Assessing cytokine supplementation to improve lymphocyte viability after cryopreservation.
Bella Circo1, Anna R. Mahr1, Alexander K. Regan1, and Paul W. Denton, PhD1
Background: A primary goal of the Denton Immunobiology Lab is improving the killing efficacy of natural killer (NK) cells. The source for our NK cells is recently collected human PBMCs
available, we desire to use cryopreserved PBMCs. However, NK cell viability within recently thawed PMBCs has been poor. To address this, we hypothesized that exogenous cytokine stimulation could allow for testing with cryopreserved cells so long as cytokine levels can be identified that lead to improved viability without altering activation state during post-thawing incubations.
Methods: Purified human IL-2 and IL-15 were obtained. We established an overlapping matrix strategy whereby recently thawed PBMC exposure to each cytokine was independently titrated at the picomolar scale. Fresh PBMC were obtained. The fresh cells were divided into a fresh control arm and into a cryopreserved arm. Fresh samples were incubated overnight in complete RPMI media (RPMI + 10% fetal bovine serum+ 1% penicillin/streptomycin). Following the overnight incubation, cell viability was determined using trypan blue exclusion. For the cryopreserved samples, multiple aliquots were frozen back identically using standard protocols. This allowed for multiple cytokine matrices to be tested with overnight incubations using recently thawed PBMCs while simultaneously controlling for inter-human variability.
Results: Current results indicate that cytokine supplementation treatment at certain titrations yields higher variability than control PBMCs. Specifically, our data suggest that IL-2 supplementation alone is leading to higher viability in comparison to IL-15 supplementation alone. Furthermore, our data are showing that the combination dosages show maximal viability when IL-2 concentrations are increased while IL-15 concentrations decreased. Data-to-date will be shown.
Conclusions: In this study, we are striving to determine whether cytokine supplementation at picomolar concentrations can lead to cellular hemostasis post-cryopreservation of PBMCs. Future work will finalize our determination of an optimal cytokine supplementation strategy and we will also explore additional supplementations (e.g., DNAase) in the media to further improve viability. Next steps will be to verify that the supplementation strategy is not leading to exogenous activation of PBMCs during this “resting” overnight culture. Once conclusions for this project are finalized, we will produce a standardized lab protocol for future experiments that involve culturing cryopreserved PBMCs.