scholarly journals Quantitative Analysis Reveals Expansion of Human Hematopoietic Repopulating Cells After Short-term Ex Vivo Culture

1997 ◽  
Vol 186 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Mickie Bhatia ◽  
Dominique Bonnet ◽  
Ursula Kapp ◽  
Jean C.Y. Wang ◽  
Barbara Murdoch ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Fold Increase ◽  
Culture Conditions ◽  
Nonobese Diabetic ◽  
Crucial Component ◽  
Cord Blood Cells ◽  
Ex Vivo Culture

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38− cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38− cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

scholarly journals Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain

Blood ◽  
2012 ◽  
Vol 119 (8) ◽  
pp. 1848-1855 ◽  
Author(s):  
Andre Larochelle ◽  
Jennifer M. Gillette ◽  
Ronan Desmond ◽  
Brian Ichwan ◽  
Amy Cantilena ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Clinical Importance ◽  
Mechanistic Explanation ◽  
Membrane Domain ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ex Vivo Culture

AbstractManipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γnull (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

2011 ◽  
Vol 337 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Daisuke Takahashi ◽  
Hiroshi Azuma ◽  
Hiromi Sakai ◽  
Keitaro Sou ◽  
Daiko Wakita ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Culture Conditions ◽  
Ex Vivo Culture

scholarly journals Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

Biology ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 748
Author(s):  
Elisa Wirthgen ◽  
Melanie Hornschuh ◽  
Ida Maria Wrobel ◽  
Christian Manteuffel ◽  
Jan Däbritz
Keyword(s):  
Blood Flow ◽  
Shear Flow ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Inflammatory Signaling ◽  
Gm Csf ◽  
Beneficial Effects ◽  
Migratory Capacity ◽  
Ex Vivo Culture ◽  
Static Conditions

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model

2006 ◽  
Vol 34 (7) ◽  
pp. 943-950 ◽  
Author(s):  
Yvette van Hensbergen ◽  
Laurus F. Schipper ◽  
Anneke Brand ◽  
Manon C. Slot ◽  
Mick Welling ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cord Blood ◽  
Blood Cells ◽  
Scid Mouse ◽  
Ex Vivo ◽  
Human Cd34 ◽  
Scid Mouse Model ◽  
Cord Blood Cells ◽  
Ex Vivo Culture

Assessment of Long-Term in vitro Multiplied Human Wharton’s Jelly-Derived Mesenchymal Stem Cells prior to Their Use in Clinical Administration

2021 ◽  
pp. 1-11
Author(s):  
Urvi Panwar ◽  
Kanchan Mishra ◽  
Parizad Patel ◽  
Sumit Bharadva ◽  
Salil Vaniawala ◽  
...  
Keyword(s):  
Telomere Length ◽  
Replicative Senescence ◽  
Ex Vivo ◽  
Wharton’S Jelly ◽  
Cellular Aging ◽  
Human Umbilical Cord ◽  
Surface Markers ◽  
Wharton's Jelly ◽  
Ex Vivo Culture

The quantity of mesenchymal stem/stromal cells (MSCs) required for a particular therapy demands their subsequent expansion through ex vivo culture. During in vitro multiplication, they undergo replicative senescence which may alter their genetic stability. Therefore, this study was aimed to analyze cellular, molecular, and chromosomal alterations in Wharton’s jelly-derived MSCs (WJ-MSCs) during their in vitro sequential passages, where WJ-MSCs were sequentially passaged up to P14 and cells were evaluated at an interval of P2, P6, P10, and P14. They were examined for their morphology, tumorigenicity, surface markers, stemness markers, DNA damage, chromosomal aberration, and telomere length. We have processed five full-term delivered human umbilical cord samples to obtain WJ-MSCs. Morphological appearance observed at initial stages was small fine spindle-shaped WJ-MSCs which were transformed to flat, long, and broader cells in later passages. The cell proliferation rate was gradually decreased after the 10th passage. WJ-MSCs have expressed stemness markers OCT-4 and NANOG, while they showed high expression of positive surface markers CD90 and CD105 and lower expression of CD34 and CD45. They were non-tumorigenic with slow cellular aging during subsequent passages. There was no chromosomal abnormality up to the 14th passage, while increase in comet score and decrease in telomere length were observed in later passages. Hence, our study suggests that early and middle passaged (less than P10) WJ-MSCs are good candidates for clinical administration for treatment.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

scholarly journals Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture

2020 ◽  
Author(s):  
JL Reading ◽  
VD Roobrouck ◽  
CM Hull ◽  
PD Becker ◽  
J Beyens ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Regulatory T Cell ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Fold Increase ◽  
Adoptive Cellular Therapy ◽  
T Cell Therapy

AbstractRecent clinical experience has demonstrated that adoptive regulatory T cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote regulatory T cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a GMP compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is linked with a distinct Treg cell-intrinsic transcriptional program, characterized by diminished levels of core exhaustion (BATF, ID2, PRDM1, LAYN, DUSP1), and quiescence (TOB1, TSC22D3) related genes, coupled to elevated expression of cell-cycle and proliferation loci (MKI67, CDK1, AURKA, AURKB). In addition, MulTreg display a unique gut homing (CCR7lo β7hi) phenotype and importantly, are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or Th1-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 TSDR demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno graft vs host disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Sign in / Sign up

Export Citation Format

Share Document