scholarly journals Establishment of a Spontaneous Stromal Microenvironment from Cord Blood Supports Human Dynamic Erythropoietic Temporal Maturation in Long-Term Serum- and Cytokine-Free 3D Cultures and Reveals a Distinct CD44hi Population

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2219-2219
Author(s):  
Susana Brito Dos Santos ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Hla Typing ◽  
High Dose ◽  
Ex Vivo Model ◽  
Erythroid Progenitor ◽  
Erythroid Precursors

Dynamic cultures which can represent physiologic erythropoiesis in vitro require a three-dimensional (3D) architecture with a supportive microenvironment and addition of erythropoietin (EPO). We have previously reported on a 3D bone marrow (BM) biomimicry using polyurethane scaffolds to expand cord blood mononuclear cells (CBMNCs) in a serum- and cytokine-free environment, without addition of dexamethasone, for 28 days (D). CBMNCs were seeded (4x106 cells/scaffold), supplemented with 10ng/mL stem cell factor (SCF; D0-D28) and 100mU/mL EPO (D7-D28), with medium exchange every 3D and exposed to a hypoxia (5%)/normoxia (20%) schedule to mimic BM oxygen gradients. Hypoxia induced rapid erythroid commitment and established an early erythroid progenitor population in the absence of EPO. Normoxia and EPO was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as crucial for endogenous cytokine production. Herein, we extended cultures to D48 using two high-dose EPO-stimulation cycles (1U/mL; D20 and D44) to enhance erythropoiesis and further define the microenvironment. Proliferation was higher after EPO pulses (p<0.05) but did not result in enhanced erythropoiesis, suggesting the absence of erythroid precursors. An allogeneic CB unit was added to "recharge" the cultured scaffolds at D39 and a new cycle of erythropoietic differentiation was initiated. Cell proliferation was 4.5-fold higher at D68, compared with that at D28. From D53-D68, CD71+CD235a+ cells were constantly produced (25-54%), corresponding with the presence of erythroid precursors supporting CFU-E and BFU-E. Erythroblastic islands were identified and maturing and enucleated reticulocytes/RBCs were abundant (19±2%; 1±0.3x106 cells) with expression of γ- and β-globin, band 3 and 4.1R RBC membrane proteins. To further evaluate the relative contributions of each CB unit to the "recharge" culture, seeded scaffolds were subjected to irradiation (or not) 48h prior to recharge. By D65, >30% of supernatant cells were CD71+/modCD235a+ and supported BFU-E and CFU-E. Proliferation in long-term cultures was attributed to the second CB unit, regardless of irradiation, as shown by HLA-typing of D68 cells; the first CBMNCs only contributed to establishment of the microenvironment. To further characterize erythroid differentiation dynamics, expression of CD44 vs CD235a was used to identify and sort three erythroid populations. Progenitors in CD44-/modCD235a- populations evolved to CD44modCD235amod, and then to CD44-/modCD235a+ cells, which constitute the most mature erythroid phenotype; CD44modCD235amod erythroid precursors supported mainly BFU-E and CFU-E. A unique CD44hiCD71mod population increased during culture, displayed myeloid progenitor morphology and only supported CFU-GM. This population did not express CD34, CD33 or CD14 but expressed c-KIT, which suggests a hematopoietic population that provides essential culture support. Further characterization of the spontaneously created microenvironment by in situ quantitative analysis of scaffold mid-sections during the 68-day culture showed varying and high dynamic expression of Nestin, STRO-1, CD146, CD68 and CD169 in separate cell populations as well as expression of RUNX2 and Osx. Osteopontin was not detected. In summary, a BM biomimicry composed of diverse stromal populations was spontaneously created in the 3D in vitro scaffold system. This microenvironment proved to effectively induce and sustain erythropoiesis to enucleation to at least D68 when supplied with a second source of CBMNCs, without addition of stroma-specific factors. A unique CD44hi immature monocyte/macrophage population was identified, which contributes to the inductive microenvironment, and distinct stages of human erythroid maturation could be identified using CD44 and CD235a. This work presents a novel and dynamic ex vivo model that can (1) recapitulate physiologic human erythropoiesis in steady-state and stress conditions, (2) capture the fetal to adult hemoglobin transition, (3) explore the direct role of oxygen on erythropoiesis, (4) assess the microenvironment relevant to erythropoiesis in the absence of serum and exogenous factors, (5) sustain long-term erythropoiesis with terminal maturation and, (6) explore the different stromal niche environments spontaneously created for the support of erythropoiesis. Disclosures Brito Dos Santos: GE Healthcare: Employment.

Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3211-3211
Author(s):  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Kazuyuki Murase ◽  
Satoshi Iyama ◽  
Tsutomu Sato ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Production System ◽  
Ex Vivo ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Human Stromal Cells

Abstract Abstract 3211 We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells. Disclosures: No relevant conflicts of interest to declare.

CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2444-2444
Author(s):  
Ewa K Zuba-Surma ◽  
Magdalena Kucia ◽  
Rui Liu ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia2007;21:297–303) These VSELs are: smaller than erythrocytes; SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−; responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin). Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. Figure Figure

scholarly journals In Vitro and Ex Vivo Activities of Minocycline and EDTA against Microorganisms Embedded in Biofilm on Catheter Surfaces

2003 ◽  
Vol 47 (11) ◽  
pp. 3580-3585 ◽  
Author(s):  
Issam Raad ◽  
Ioannis Chatzinikolaou ◽  
Gassan Chaiban ◽  
Hend Hanna ◽  
Ray Hachem ◽  
...  
Keyword(s):  
Low Dose ◽  
Ex Vivo ◽  
Bacterial Colonization ◽  
Rabbit Model ◽  
Confocal Laser ◽  
High Dose ◽  
High Concentration ◽  
Ex Vivo Model ◽  
Highly Active

ABSTRACT Minocycline-EDTA (M-EDTA) flush solution has been shown to prevent catheter-related infection and colonization in a rabbit model and in hemodialysis patients. We undertook this study in order to determine the activities of M-EDTA against organisms embedded in fresh biofilm (in vitro) and mature biofilm (ex vivo). For the experiment with the in vitro model, a modified Robbin’s device (MRD) was used whereby 25 catheter segments were flushed for 18 h with 106 CFU of biofilm-producing Staphylococcus epidermidis, Staphyloccocus aureus, and Candida albicans per ml. Subsequently, each of the catheter segments was incubated in one of the following solutions: (i) streptokinase, (ii) heparin, (iii) broth alone, (iv) vancomycin, (v) vancomycin-heparin, (vi) EDTA, (vii) minocycline (high-dose alternating with low-dose), or (viii) M-EDTA (low-dose minocycline alternating with high-dose minocycline were used to study the additive and synergistic activities of M-EDTA). All segments were cultured quantitatively by scrape sonication. For the experiment with the ex vivo model, 54 catheter tip segments removed from patients and colonized with bacterial organisms by roll plate were longitudinally cut into two equal segments and exposed to either saline, heparin, EDTA, or M-EDTA (with high-dose minocycline). Subsequently, all segments were examined by confocal laser electron microscopy. In the in vitro MRD model, M-EDTA (with a low concentration of minocycline) was significantly more effective than any other agent in reducing colonization of S. epidermidis, S. aureus, and C. albicans (P < 0.01). M-EDTA (with a high concentration of minocycline) eradicated all staphylococcal and C. albicans organisms embedded in the biofilm. In the ex vivo model, M-EDTA (with a high concentration of minocycline) reduced bacterial colonization more frequently than EDTA or heparin (P < 0.01). We concluded that M-EDTA is highly active in eradicating microorganisms embedded in fresh and mature biofilm adhering to catheter surfaces.

scholarly journals A Murine Model for Human Cord Blood Transplantation: Near-Term Fetal and Neonatal Peripheral Blood Cells Can Achieve Long-Term Bone Marrow Engraftment in Sublethally Irradiated Adult Recipients

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1089-1099 ◽  
Author(s):  
Andromachi Scaradavou ◽  
Luis Isola ◽  
Pablo Rubinstein ◽  
Yelena Galperin ◽  
Vesna Najfeld ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Blood Transplantation ◽  
Near Term ◽  
Ex Vivo Culture

Abstract The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34+ cord blood cells

2000 ◽  
Vol 28 (12) ◽  
pp. 1470-1480 ◽  
Author(s):  
Ladan Kobari ◽  
Françoise Pflumio ◽  
Marie-Catherine Giarratana ◽  
Xiaxin Li ◽  
Monique Titeux ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Human Cd34 ◽  
Cord Blood Cells

In vitro And In vivo Evidences for the long-term multilineage (Myeloid, b, nk & t) Reconstitution capacity of Ex vivo Expanded human CD34+ cord blood cells

2000 ◽  
Vol 28 (7) ◽  
pp. 47-48
Author(s):  
L. Douay ◽  
L. Kobari ◽  
F. Pflumio ◽  
M.C. Giarratana ◽  
X. Li ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Human Cd34 ◽  
Cord Blood Cells

scholarly journals The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

Blood ◽  
2020 ◽  
Vol 136 (21) ◽  
pp. 2410-2415 ◽  
Author(s):  
Peng Hua ◽  
Joanna Hester ◽  
George Adigbli ◽  
Rong Li ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Bet Inhibitor ◽  
Functional Studies

Abstract Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

scholarly journals Toxicological profile of umbilical cord blood-derived small extracellular vesicles

2021 ◽  
Author(s):  
Silvia C Rodrigues ◽  
Renato M S Cardoso ◽  
Claudia F Gomes ◽  
Filipe V Duarte ◽  
Patricia C Freire ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
High Dose ◽  
Cell Therapies ◽  
Cell Counts ◽  
Blood Mononuclear Cells

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1x10^11 particles/ml had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts or human umbilical vein endothelial cells. DiR-labeled sEV, injected IV for four weeks in healthy rats, were detected in clearance organs, particularly kidneys, spleen and liver, similarly to control dye. Moreover, repeated administrations during six and twelve weeks of up to 1x10^10 total particles of sEV-dye were well tolerated, with no changes in general hematological cell counts, or kidney and liver toxicity markers. Importantly, unlabeled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in heart, kidney, lung, spleen, or liver tissue. In sum, our data shows that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

scholarly journals Toxicological Profile of Umbilical Cord Blood-Derived Small Extracellular Vesicles

Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 647 ◽  
Author(s):  
Silvia C. Rodrigues ◽  
Renato M. S. Cardoso ◽  
Claudia F. Gomes ◽  
Filipe V. Duarte ◽  
Patricia C. Freire ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
High Dose ◽  
Cell Therapies ◽  
Cell Counts ◽  
Blood Mononuclear Cells

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling, and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high-dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution, and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1 × 1011 particles/mL had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts, or human umbilical vein endothelial cells. DiR-labelled sEV, injected intravenously for four weeks in healthy rats, were detected in clearance organs, particularly the kidneys, spleen, and liver, similarly to control dye. Moreover, repeated administrations for six and twelve weeks of up to 1 × 1010 total particles of sEV dye were well-tolerated, with no changes in general haematological cell counts, or kidney and liver toxicity markers. More importantly, unlabelled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in the heart, kidney, lung, spleen, or liver tissue. In sum, our data show that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

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