scholarly journals Thawed and Washed Apheresis Products Keep an Excellent CD34+ Cells Viability for 24 Hours Using Either Voluven or Normal Saline Plus Albumin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5738-5738
Author(s):  
Miguel Blanquer Blanquer ◽  
Carmen Alguero ◽  
Pilar Menchon ◽  
Assumpta Ferrer ◽  
Pilar Martínez Avilés ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Normal Saline ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Time Points ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
The Mean

Abstract Cryopreservation of products rich in progenitor cells is mandatory for the feasibility of autologous hematopietic progenitor cells transplants. The most used product nowadays is the apheresis of mobilized peripheral blood mononuclear cells. However, cryopreservation implies the use of cryoprotectant molecules, such as DMSO, that can be toxic for the patients. Moreover, the thawing of the product goes with some unavoidable cell death and liberation of cytoplasmic content such as cytokines to the medium. And so, adverse reactions during the product infusion are not infrequent. Our group has demonstrated that washing the thawed apheresis products both with Voluven or with normal saline plus 5% albumin (NSA) is able to almost completely avoid infusion reactions without losing CD34+ cells. Here we wanted to know whether it also had the benefit of extending CD34+ viability for 24 hours after washing. METHODS: We thawed 3 spare peripheral blood mononuclear cells apheresis products that had been cryopreserved with 9%DMSO. Ten mL of each product were separated and the remaining volume was splitted in 2 bags to be washed either with Voluven or NSA. Sepax 2 smartwash automatic program was used to wash the cells. The washed cells were stored at 4ºC and a sample was taken and immediately analyzed at 0h, 1h, 2h, 4h and 24h. At the same time points 10 mL of each bag were separated and kept 30 min. at room temperature (RT) before being analyzed. A blood count was performed on all the samples. Flow cytometry was used to measure CD45+ and CD34+ cells viability by 7AAD staining. RESULTS: The mean CD45+ cells viability was 69% after thawing the cells ,81% immediately after washing them with Voluven, and 81%, 80%, 79% and 73% 1h, 2h, 4h and 24h after the wash. When using NSA the mean CD45+ viability was 79%, 92%, 93%, 92%, 93% and 87% at the same time points. When the samples were kept for 30 additional minutes at RT, the mean CD45+ viability was 78%, 81%, 84%, 82%, 84% and 81% with Voluven, and 80%, 92%, 94%, 91%, 95% and 86% with NSA. Regarding CD34+ cells, when washed with Voluven the mean viability was 46%, 87%, 87%, 92%, 83% and 69%, while it was 61%, 91%, 91%, 92%, 89% and 84% when NSA was used. The mean CD34+ cells viability results after 30' at RT for each time point were 79%, 90%, 82%, 91%, 82% and 81% with Voluven and 85%, 90%, 90%, 94%, 93% and 86% with NSA. Moreover the mean viable CD34+ cells recovery at 24h was 90.94% for Voluven and 87,88% for NSA. CONCLUSIONS: We have obtained an excellent stability of the CD34+ cells viability for 24h after washing the mobilized mononuclear cells apheresis products both with Voluven and with NSA when the products are stored at 4ºC. Moreover, the viability is not affected when the cells are kept for an additional 30' at RT. The viable CD34+ cells loss at 24h was scarce. Disclosures Blanquer Blanquer: Pfizer: Research Funding.

CD34+ progenitor cells mobilized by natalizumab are not a relevant reservoir for JC virus

2010 ◽  
Vol 17 (2) ◽  
pp. 151-156 ◽  
Author(s):  
Clemens Warnke ◽  
Vsevolod Smolianov ◽  
Thomas Dehmel ◽  
Marcel Andrée ◽  
Hartmut Hengel ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Jc Virus ◽  
Viral Dna ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
Natalizumab Treatment

Background: Progressive multifocal leukoencephalopathy (PML) is associated with natalizumab treatment in patients with multiple sclerosis (MS). It has been hypothesized that natalizumab mobilizes JC virus (JCV)-infected haematopoietic progenitor cells mediating viraemia and subsequently this disease. Objective: The objective of this study was to investigate peripheral haematopoietic progenitor cells for evidence of JCV DNA in MS patients treated with natalizumab. Methods: We assessed JCV and cytomegalovirus (CMV) DNA in magnetically separated CD34+ haematopoietic progenitor cells, peripheral blood mononuclear cells and plasma of 67 natalizumab-treated patients with MS and six PML patients. Results: Viral DNA was not detectable in CD34+ haematopoietic progenitor or peripheral blood mononuclear cells from any sample. Two plasma samples from patients with MS while undergoing natalizumab treatment were JCV-positive. In one case clinically manifest PML developed 8 months thereafter. Conclusions: Our findings do not support the hypothesis that natalizumab mobilizes JC virus-infected CD34+ cells from the bone marrow mediating JC viraemia. Notably, JC viraemia was detected in one patient with MS prior to developing clinical PML. This warrants further study.

scholarly journals Integrated microRNA and mRNA Gene Expression in Peripheral Blood Mononuclear Cells in Response to Acute Psychosocial Stress: A Repeated-Measures Within-Subject Pilot Study

2021 ◽  
Author(s):  
Magdalena Maria Jurkiewicz ◽  
Anett Müller-Alcazar ◽  
Dirk Moser ◽  
Indralatha Jayatilaka ◽  
Anatoly Mikhailik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Psychosocial Stress ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Time Points ◽  
Blood Mononuclear Cells ◽  
The Impact ◽  
Acute Psychosocial Stress

Abstract Objective: The impact of psychosocial stress on a variety of negative health outcomes is well documented, with current research efforts directed at possible mechanisms. Here, we focused on a potential mechanism involving differential expression of mRNA and microRNA in response to acute psychosocial stress. We utilized a validated behavioral paradigm, the Trier Social Stress Test (TSST), to induce acute psychosocial stress in a cohort of volunteers. Stress reactivity was assessed repeatedly during the TSST using saliva samples that were analyzed for levels of cortisol. Peripheral blood mononuclear cells were extracted from blood drawn at baseline and at two time points following the stress paradigm. Total RNA was extracted, and mRNA and microRNA microarrays were utilized to assess within-subject changes in gene expression between baseline and the two post-stressor time points. Results: For microarray gene expression analysis, we focused on 12 participants who showed a robust cortisol response to the task, as an indicator of robust HPA-axis activation. We discovered a set of mRNAs and miRNAs that exhibited dynamic expression change in response to the TSST in peripheral blood mononuclear cells, further characterizing the link between psychosocial stress and cellular response mechanisms.

Identification Of Specific Hematopoietic Populations Present In Peripheral Blood Mononuclear Cells That Increase Erythroid Output Directly Or Through Feeder/Stromal Cell-Like Properties

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3414-3414
Author(s):  
Esther Heideveld ◽  
Valentina Tirelli ◽  
Francesca Masiello ◽  
Fatemehsadat Esteghamat ◽  
Nurcan Yagci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Cell Contact ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Abstract Hematopoietic development occurs in defined niches that ensure specific interactions and cross-talk with the surrounding stromal cells and different hematopoietic cells themselves. For instance, erythropoiesis occurs on the macrophage island within the bone marrow and the central macrophage is believed to regulate pro-erythroblast differentiation, the final stages of enucleation and reticulocyte maturation. We have observed that the expansion of erythroblasts from total peripheral blood mononuclear cells is increased compared to CD34+ Hematopoietic Stem/Progenitor Cells (HS(P)C) isolated from the same amount of blood [van den Akker, Haematologica, 2010]. This suggests i) the presence of CD34-cells that contribute to erythropoiesis and/or ii) that cell-cell contact or specific secreted growth factors by “helper” cells in these cultures can regulate hematopoiesis/erythropoiesis to increase erythroblast yield. Identifying the specific population(s) underlying the increased erythroid yield and understanding their way of action and regulatory mechanism during HSC differentiation and erythropoiesis is not only important to improve erythroblast culture conditions but may also provide clues to the function of hematopoietic effector cells in the various HS(P)C/erythroblast niches. Using specific lineage depletion (among which CD3 and CD14) we have identified and quantified various human erythroid and non-erythroid CD34+ and CD34- populations on the basis of CD36 co-expression in peripheral blood mononuclear cells (PBMC). Erythroid outgrowth from these CD34- populations and CD34+ populations and their contribution to the total erythroid yield from PBMC was assessed. Interestingly, total erythroid yield from the individual sorted populations did not reach the erythroid yield obtained from total PBMC. We hypothesized that support/feeder cells present in total PBMC are positively influencing in vitro erythropoiesis. In agreement with this, PBMC immuno-depletion of specific hematopoietic cell types identified CD14 cells (monocyte/macrophages) and to a lesser extend CD3 cells (lymphocytes) to be also partly responsible for the increased erythroblast yield. Compared to HS(P)C alone, co-culture of CD14 cells and HS(P)C isolated from PBMC resulted in a 5-10 times increase in CD71high/CD235med erythroblasts. Conditioned medium of CD14 cells as well as transwell experiments reconstituted the effect of the HS(P)C-CD14 co-cultures to 70%-80%, indicating that cell-cell contact plays a minor role. CD14 cells could elicit their effect at different stages during HSPC/HSC differentiation to erythroblasts. Co-culture of CD14 cells with pro-erythroblasts did not increase the cellular yield or proliferation rate. In contrast, two days of CD14 co-culture with CD34+ cells results in a 5 fold increase of total colony forming units without altering the colony lineage dynamics. In agreement with this a 5 fold increase in CD34+ cells was observed. These results indicate that CD14 cells elicit their effect on early hematopoietic progenitors but not on the erythroblast population. The results predict that depletion of CD14+ cells from PBMC should result in a decrease in the total number of CD34+cells. Indeed, we observed a 2 fold decrease of specifically HS(P)Cs and MEPs after two days of culture in PBMCs depleted for CD14 cells. Taken together our data i) identify previously unrecognized erythroid and non erythroid CD34- and CD34+ populations in peripheral blood that contribute to erythroid yield from total PBMC and ii) indicate modulation of HS(P)C outgrowth by specific hematopioietic effector cells present in peripheral blood that can also be found near specific hematopoietic niches in the bone marrow. The involvement of CD3 and CD14 immune cells suggests that HS(P)C and erythropoiesis may be modulated by immune-responses. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 958-970 ◽  
Author(s):  
CE Sandstrom ◽  
JG Bender ◽  
ET Papoutsakis ◽  
WM Miller
Keyword(s):  
Cell Cultures ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Similar Distribution ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

scholarly journals Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model

2018 ◽  
Vol 27 (7) ◽  
pp. 1068-1079 ◽  
Author(s):  
Makiko Kado ◽  
Rica Tanaka ◽  
Kayo Arita ◽  
Kayoko Okada ◽  
Rie Ito-Hirano ◽  
...  
Keyword(s):  
Wound Healing ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Skin Defect ◽  
Endothelial Progenitor ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.

Leukoreduction System Chambers as a Source of Viable Human Peripheral Blood Mononuclear Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4912-4912
Author(s):  
Sinyoung Kim ◽  
Han-Soo Kim ◽  
Yangsoon Lee ◽  
Jaewoo Song ◽  
Hyun Ok Kim ◽  
...  
Keyword(s):  
Cell Count ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Buffy Coat ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
The Mean

Abstract Many blood banks are using apheresis machines to collect blood components such as platelets (PLTs), RBCs, or plasma. Especially, leukoreduced plateletpheresis using apheresis instrument (Trima Accel, Gambro BCT, Lakewood, CO) provided subsidiary cell products retained in leukoreduction system (LRS) chamber that was originally discarded. The LRS chamber is a conical-shaped chamber that uses saturated, fluidized, particle bed filtration technology to remove WBCs from PLTs. In the current study, a total of 24 LRS chambers from different donors were investigated to determine it would be a valuable source of viable human peripheral blood mononuclear cells (MNCs). The proportions of CD3+, CD19+, CD16+/CD56+, CD14+, CD45+ cells, and absolute CD34+ cell count within the LRS chambers were determined by flow cytometry. Dendritic cells (DCs) were generated from the immunomagnetically purified CD14+ cells from LRS chamber and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the LRS chamber, the total number of WBC count was 1.1 × 109 ± 0.3 × 109 and the mean percentage of MNCs was 80.6 ± 13.1%. The mean proportion of T cells, B cells, NK cells, CD14+ monocytes among CD45+ cells was 54.3 ± 11.5%, 6.4 ± 3.1%, 14.6 ± 3.9%, 12.9 ± 7.5%, respectively. Total absolute CD34+ cell count in LRS chamber was 0.95 × 106 ± 0.65 × 106. Also, we could demonstrate CD14+ cells isolated from LRS chamber was capable of differentiating into functionally mature DCs in vitro. LRS chambers are a valuable and convenient source of viable human peripheral blood mononuclear cell population and could replace standard buffy coat preparations for research applications.

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