Evaluation of a Xeno-Free Protocol for Long-Term Cryopreservation of Cord Blood Cells

Mairhofer, Mario; Schulz, Julia; Parth, Michela; Beer, U.; Zimmermann, Heiko; Kolbus, Andrea

doi:10.3727/096368912x657396

Cited by 3 publications

(3 citation statements)

References 31 publications

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“…Isolated PBMCs were frozen in cryomedium IBMT I (Fraunhofer IBMT, Sulzbach, Germany) 35 with a final concentration of 1 × 10 7 cells/mL in 1-mL aliquots. The samples were transferred into precooled (+4°C), freezing isopropanol containers (Mr. Frosty ™ ; Thermo Fisher Scientific, Dreieich, Germany) where the cells were frozen at a controlled cooling rate of −1°C/min from +4°C to −80°C.…”

Section: Methodsmentioning

confidence: 99%

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

Angel

et al. 2016

Biopreservation and Biobanking

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Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

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Section: Methodsmentioning

confidence: 99%

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

Angel

et al. 2016

Biopreservation and Biobanking

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“…Cord blood samples from healthy human donors were collected from full-term deliveries; mononuclear cells were isolated as described (Mairhofer et al, 2013) and stored in liquid nitrogen. Hematopoietic precursors were separated from defrosted mononuclear cells using CD34 magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec), the cell numbers of viable CD34 þ cells was determined using a modified ISHAGE gating strategy (Mairhofer et al, 2013) and dead cells were excluded with DAPI (BD Biosciences, Franklin Lakes, NJ). CD34 þ hematopoietic precursors were cultured for 3 days under expansion conditions using X-Vivo 15 (Lonza) containing penicillin (100 U/l)/streptomycin (100 mg/ml) and supplemented with thrombopoetin (50 ng/ml), SCF (50 ng/ml), and Flt3L (50 ng/ml; all Peprotech).…”

Section: Isolation and Differentiation Of Cord Blood-derived Cd34 D Hematopoietic Precursorsmentioning

confidence: 99%

The Reticulum-Associated Protein RTN1A Specifically Identifies Human Dendritic Cells

Gschwandtner

Kienzl

Tajpara

et al. 2018

Journal of Investigative Dermatology

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RTN1 is an endoplasmic reticulum-associated protein that was initially identified in neuronal tissues. Here we show that the main isoform RTN1A is a marker for dendritic cells. In the skin, HLA-DRCD1aCD207CD11c Langerhans cells were the only cells in the epidermis, and HLA-DRCD11c dendritic cells were the main cells in the dermis, expressing this protein. RTN1A dendritic cells were also found in gingiva, trachea, tonsil, thymus, and peripheral blood. During differentiation of MUTZ-3 cells into Langerhans cells, expression of RTN1A mRNA and protein preceded established Langerhans cell markers CD1a and CD207, and RTN1A protein partially co-localized with the endoplasmic reticulum marker protein disulfide isomerase. In line with this observation, we found that RTN1A was expressed by around 80% of Langerhans cell precursors in human embryonic skin. Our findings show that RTN1A is a marker for cells of the dendritic lineage, including Langerhans cells and dermal dendritic cells. This unexpected finding will serve as a starting point for the elucidation of the, until now, elusive functional roles of RTN1A in both the immune and the nervous system.

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“…The collection was approved by the local ethics committee and conducted in accordance with the declaration of Helsinki Principles. Mononuclear cells were isolated as described and stored in liquid nitrogen (Mairhofer et al, 2013). After thawing, CD34 + haematopoietic precursors were separated from cord blood mononuclear cells by positive immunoselection (CD34 + MicroBead Kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and the number of isolated, viable CD34 + cells was determined using a modified ISHAGE gating strategy (Mairhofer et al, 2013).…”

Section: Generation Of Cord Blood-derived Cd34mentioning

confidence: 99%

Human embryonic epidermis contains a diverse Langerhans cell precursor pool

et al. 2014

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Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207-cord bloodderived haematopoietic precursor cells resembling foetal LC precursors but not CD14 + CD16-blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14 -haematopoietic precursors possess a much higher differentiation potential than CD14 + precursor cells.

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Evaluation of a Xeno-Free Protocol for Long-Term Cryopreservation of Cord Blood Cells

Cited by 3 publications

References 31 publications

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

The Reticulum-Associated Protein RTN1A Specifically Identifies Human Dendritic Cells

Human embryonic epidermis contains a diverse Langerhans cell precursor pool

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