Dataset Information

Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials.

ABSTRACT: BACKGROUND:Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC. PATIENTS/METHODS:EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45- CD34+CD133+/-VEGFR2+/- and EC were gated for CD45-CD146+CD34+/-VEGFR2+/-. RESULTS:Cryopreservation for 7 days at -80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at -65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45- CD34+CD133+VEGFR2+) were enumerated as 0.2 ± 0.06%, late EPC(CD45-CD146+CD34+VEGFR2+) as 0.6 ± 0.1% and mature EC (CD45-CD146+CD34-VEGFR2+) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at -80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant. CONCLUSIONS:Our data indicate that cryopreservation at -80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.

SUBMITTER: Bogoslovsky T

PROVIDER: S-EPMC4193669 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials.

Bogoslovsky T T Wang D D Maric D D Scattergood-Keepper L L Spatz M M Auh S S Hallenbeck J J

Journal of blood disorders & transfusion 20130901 5

<h4>Background</h4>Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC.<h4>Patients/methods</h4>EPC and EC wer ...[more]

PMID: 25309814

Similar Datasets

Project description:Human pluripotent stem cells can be differentiated into midbrain dopaminergic (mDA) neurons by directing cells through a floor plate progenitor stage. The developmental identity of mDA neurons produced using floor plate protocols is similar to substantia nigra neurons, and this has improved the ability to model Parkinson's disease (PD) in a dish. Combined with the unlimited growth potential of pluripotent stem cells, mDA neural progenitor cell production can provide a scalable source of human dopaminergic (DA) neurons for diverse applications. However, due to the complexity and length of the protocols and inherent differences between cell lines, considerable variability of the final population of neurons is often observed. One solution to this problem is to cryopreserve committed mDA neural progenitor cells in a ready-to-use format. Creating a bank of cryopreserved mDA neural progenitor cells poised for neuronal differentiation could significantly improve reproducibility and facilitate collaborations. Here we have compared six (6) different commercial cryopreservation media and different freezing conditions for mDA neural progenitor cells differentiated from human embryonic stem cell (hESC) lines. Significant differences in cell recovery were observed at 24 h post-thawing, but no differences were observed immediately upon thawing. The presence of ROCK inhibitors improved cell recovery at 24 h for all cryopreservation media tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Importantly, cryopreservation of mDA neural progenitor cells did not alter their potential to resume differentiation into mDA neurons. Banks of cryopreserved committed mDA neural progenitor cells provide a method to generate human DA neurons with reduced batch-to-batch variability, and establish a mechanism to share lineage-primed cells for collaborative research.

Project description:The blood-brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.

Dataset Information

Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials.

Publications

Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets