Cargando…

Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies

Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permut...

Descripción completa

Detalles Bibliográficos
Autores principales: Ramachandran, Hari, Laux, Jessica, Moldovan, Ioana, Caspell, Richard, Lehmann, Paul V., Subbramanian, Ramu A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901099/
https://www.ncbi.nlm.nih.gov/pubmed/24710478
http://dx.doi.org/10.3390/cells1030313
_version_ 1782300798357602304
author Ramachandran, Hari
Laux, Jessica
Moldovan, Ioana
Caspell, Richard
Lehmann, Paul V.
Subbramanian, Ramu A.
author_facet Ramachandran, Hari
Laux, Jessica
Moldovan, Ioana
Caspell, Richard
Lehmann, Paul V.
Subbramanian, Ramu A.
author_sort Ramachandran, Hari
collection PubMed
description Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.
format Online
Article
Text
id pubmed-3901099
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-39010992014-04-07 Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies Ramachandran, Hari Laux, Jessica Moldovan, Ioana Caspell, Richard Lehmann, Paul V. Subbramanian, Ramu A. Cells Article Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments. MDPI 2012-07-25 /pmc/articles/PMC3901099/ /pubmed/24710478 http://dx.doi.org/10.3390/cells1030313 Text en © 2012 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Ramachandran, Hari
Laux, Jessica
Moldovan, Ioana
Caspell, Richard
Lehmann, Paul V.
Subbramanian, Ramu A.
Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title_full Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title_fullStr Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title_full_unstemmed Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title_short Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies
title_sort optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901099/
https://www.ncbi.nlm.nih.gov/pubmed/24710478
http://dx.doi.org/10.3390/cells1030313
work_keys_str_mv AT ramachandranhari optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies
AT lauxjessica optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies
AT moldovanioana optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies
AT caspellrichard optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies
AT lehmannpaulv optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies
AT subbramanianramua optimalthawingofcryopreservedperipheralbloodmononuclearcellsforuseinhighthroughputhumanimmunemonitoringstudies