Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.

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The Diverse Potential of Gluten from Different Durum Wheat Varieties in Triggering Celiac Disease: A Multilevel In Vitro, Ex Vivo and In Vivo Approach

Nutrients ◽

10.3390/nu12113566 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3566

Author(s):

Federica Gaiani ◽

Sara Graziano ◽

Fatma Boukid ◽

Barbara Prandi ◽

Lorena Bottarelli ◽

...

Keyword(s):

Immune Response ◽

Celiac Disease ◽

Durum Wheat ◽

Ex Vivo ◽

Diet Group ◽

Control Group ◽

Wheat Varieties ◽

Before And After

The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties (n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects (n = 46) were: 19 healthy subjects included in the control group; 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response.

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Neutralization of Enoxaparine-lnduced Bleeding by Protamine Sulfate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645207 ◽

1990 ◽

Vol 63 (02) ◽

pp. 271-274 ◽

Cited By ~ 70

Author(s):

J Van Ryn-McKenna ◽

L Cai ◽

F A Ofosu ◽

J Hirsh ◽

M R Buchanan

Keyword(s):

Blood Loss ◽

Unfractionated Heparin ◽

Ex Vivo ◽

Factor Xa ◽

Protamine Sulfate ◽

Low Molecular Weight Heparins ◽

Before And After ◽

Made In

SummaryIt has been suggested that protamine sulfate is a poor antidote for the bleeding side-effeets of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effeets of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin ± protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effeets of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.

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LIPID NANOPARTICLES FOR TRANSDERMAL DELIVERY OF CELECOXIB: AN IN VITRO AND IN VIVO INVESTIGATION

INDIAN DRUGS ◽

10.53879/id.56.08.11694 ◽

2019 ◽

Vol 56 (08) ◽

pp. 38-48

Author(s):

S. V Shinde ◽

S Nikam ◽

P Raut ◽

M. K. Ghag ◽

Keyword(s):

Solid Lipid Nanoparticles ◽

Ex Vivo ◽

Research Work ◽

Lipid Nanoparticles ◽

Inflammatory Activity ◽

Promising Alternative ◽

Solid Lipid ◽

Anti Inflammatory

In the present research work, celecoxib (CXB) loaded solid lipid nanoparticles (SLNs) were prepared using the probe sonication method, wherein Glyceryl monostearate and Tween 80 were used as solid lipid and surfactant, respectively. To obtain the statistically optimized batch, 32 factorial design was applied. The optimized batch was characterized physicochemically and evaluated through DSC, SEM and XRD studies. The mean particle size of the optimized batch was found to be 135.41± 0.24 nm with a mean % entrapment efficiency of 80 ± 1.69%. The optimized batch was further lyophilized and dispersed into 1% w/v Carbopol 934P to form a gel. Prepared gel was further evaluated for in vitro drug release, occlusivity, ex vivo permeability, local toxicity, in vivo anti-inflammatory activity and accelerated stability study. The study resulted in stable, safe and prolonged anti-inflammatory activity with quick onset of action. Hence, celecoxib loaded solid lipid nanoparticles can be considered as promising alternative to conventional topical systems.

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A Comprehensive High Resolution Scrna-Seq Analytical Platform for Characterizing Human Hematopoietic Stem/Progenitor Cells States in Ex Vivo Drug Products

Blood ◽

10.1182/blood-2020-141057 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Cristina Baricordi ◽

Mariana Loperfido ◽

Aimin Yan ◽

Luigi Barbarossa ◽

Mercedes Segura ◽

...

Keyword(s):

Genetic Modification ◽

Ex Vivo ◽

Single Cells ◽

Publicly Traded ◽

Dynamic Changes ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Before And After

Human hematopoietic/stem progenitor CD34+ cells (HSPCs) are the core components of ex vivo lentiviral gene therapy (GT) for the treatment of rare monogenic diseases. Assessment of CD34+ composition before and after genetic modification as well as in vivo upon transplantation in the patient is a critical parameter to establish the efficacy of GT. We recently showed how the currently available immunophenotyping markers for the identification of HSPC subpopulations do not allow a clear-cut identification of the diversity of cell states within the CD34+ compartment. Furthermore, due to the instability of such markers upon in vitro manipulation, immunophenotyping does not offer a reliable assay to measure the HSPC content within the CD34+ Drug Product (DP). We have combined immunophenotyping, FACS sorting, and selective in vitro manipulation conditions with single-cell RNA-Sequencing (scRNA-Seq) capturing a picture of unprecedent clarity of CD34+ cells dynamics before and after expansion and genetic modification. Using the 10X Chromium System we firstly generated a comprehensive baseline map of CD34+ cell states (total of 77,692 single cells) from the 3 main sources of human HSPCs: bone marrow (BM), mobilized peripheral blood (G-CSF+plerixafor mobilization, MPB) and cord blood (CB). The combination of unsupervised clustering and expression of lineage-associated gene signatures allowed the identification of 11 cell states within these bulk CD34+ populations, and unveiled key differences in their composition depending on different cells' origin. We then investigated the dynamic changes of MPB CD34+ cells, the most advanced and clinically relevant HSPC cell source, upon ex vivo manipulation. To this aim we cultured bulk CD34+ cells and 7 FACS-sorted CD34+ subpopulations independently for 40 hours in presence of SCF, IL3, FLT3-L and TPO. Classical immunophenotyping of the CD34+ population before and after culture showed a substantial enrichment of cells with a CD34+CD38- profile suggesting a selective in vitro maintenance and expansion of primitive progenitors. However, immunophenotyping of individually cultured HSPC subsets revealed that the major factor contributing to this observation is instead the progressive loss of CD38 expression by CD38+ committed progenitors. Thus, the currently available CD34+ immunophenotyping characterization is not designed to provide an accurate assessment of the true DP composition before infusion into patients. By means of scRNA-Seq analysis of 81,126 single cells we were able to identify with high granularity the changes in cell state of each HSPC subset during culture. Firstly, the combined scRNA-seq analysis on sorted HSPC subsets at baseline allowed to draw the highest resolution map of MPB CD34+ cell states available to date (total of 24,736 single cells) and allowed the identification of 7 novel transcriptional states which are independent from the original cell fractionation based on the known CD34+ surface markers. Using these transcriptional signatures we interrogated the dynamic changes of each HSPC subset looking for transcriptional divergences/similarities between the baseline and the end of culture cell states. We could identify the trajectories of each subpopulation towards different hematopoietic differentiation stages upon in vitro manipulation. As expected HSCs and MPPs were the only subsets which maintained after culture a fraction of cells retaining the original primitive transcriptional signature. We believe that in this way we could quantify with unprecedented accuracy the putative fraction of cells in the DP that preserved a multipotent long-term potential vs the one that progressed towards myeloid differentiation identifying in the process variable degrees of cell maturation. We are currently exploring specific molecular signatures within the HSC compartment, which remained stable before and after culture, for the identification of novel more reliable markers of human HSC to be validated in vitro and in vivo. Overall our analytical platform provides the basis for unravelling and comparing the impact of multiple conditions of cell cultures and gene modification on the HSPC DP. To our knowledge this constitutes the most advanced suite for the comprehensive characterization of CD34+ cells states, with potential applications spanning manufacturing, pre-clinical and clinical stages. CB and ML: equal contribution Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Loperfido:AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Yan:AVROBIO Inc: Current Employment. Barbarossa:AVROBIO Inc: Other: Trainee. Segura:AVROBIO Inc: Current Employment. Golipour:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company. Mason:AVROBIO Inc: Current Employment, Current equity holder in private company. Biasco:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company.

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Platelet Aggregation and In Vivo Shear Forces

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642380 ◽

1994 ◽

Vol 71 (01) ◽

pp. 026-031 ◽

Cited By ~ 11

Author(s):

Madeleine M Mazeaud ◽

Jaime Levenson ◽

Kim Hanh Le Quan Sang ◽

Alain Simon ◽

Marie-Aude Devynck

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Platelet Reactivity ◽

Velocity Shear ◽

Direct And Indirect Effects ◽

Shear Forces ◽

Before And After

SummaryHaemodynamic shear forces have been reported to exert direct and indirect effects on platelet reactivity. In vitro, they activate platelets leading to spontaneous or facilitated aggregation. In vivo, they stimulate the production of endothelium-derived antiaggregatory agents. This study was designed to evaluate in hypertensive patients, before and after antihypertensive treatment, the possible role of these haemodynamic forces, determined at the brachial artery level on the ex vivo platelet aggregatory response to ADP and collagen. Platelet reactivity, evaluated by EC50 for ADP and collagen, was found to be related to blood velocity, shear rate and shear stress (p <0.01 for each). These inverse correlations of platelet aggregation with stress levels did not depend on age, body mass index, mean blood pressure, serum cholesterol and triglycerides or haematocrit. They were also independent of platelet cytosolic Ca2+ and cyclic AMP.The changes in shear forces and in aggregatory responses to ADP and collagen induced by nitrendipine treatment for 6 months remained negatively correlated, confirming the relationships existing between haemodynamic shear forces and platelet reactivity.These results indicate that the shear antiaggregant effects, likely mediated by tlow-dependent endothelium-derived factors, prevail over its direct platelet aggregating effects.

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Modulation of arabinosylcytosine metabolism by arabinosyl-2- fluoroadenine in lymphocytes from patients with chronic lymphocytic leukemia: implications for combination therapy

Blood ◽

10.1182/blood.v74.6.2070.2070 ◽

1989 ◽

Vol 74 (6) ◽

pp. 2070-2075 ◽

Cited By ~ 44

Author(s):

V Gandhi ◽

B Nowak ◽

MJ Keating ◽

W Plunkett

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Ex Vivo ◽

K562 Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Median Increase ◽

Cellular Concentration ◽

Before And After

Abstract Our previous studies indicated that K562 cells loaded with arabinosyl-2- fluoroadenine 5′-triphosphate (F-ara-ATP) accumulated arabinosylcytosine 5′-triphosphate (ara-CTP) at a threefold higher rate compared to the control cells. In the present study lymphocytes were obtained from patients with chronic lymphocytic leukemia before and after F-ara-A monophosphate therapy. The rate of ara-CTP accumulation after in vitro ara-C incubation was compared in lymphocytes obtained prior to therapy without any other manipulation, after ex vivo F-ara- ATP (100 mumol/L) treatment, and after in vivo F-ara-A monophosphate therapy. Lymphocytes showed a 2.2-fold (n = 23) and 1.7-fold (n = 23) median increase in the cellular concentration of ara-CTP after an ex vivo incubation with 100 mumol/L F-ara-A and 20 to 24 hours after the first dose (25 or 30 mg/m2) of F-ara-A monophosphate in vivo treatment, respectively. Although the rates of F-ara-ATP and ara-CTP accumulation varied among patients, a relationship was observed in individuals between the cellular concentration of F-ara-ATP at the beginning of the ara-C incubation and ara-CTP accumulation. These studies strongly suggest that a protocol designed to administer F-ara-A monophosphate prior to ara-C infusion will augment ara-CTP accumulation by leukemia cells.

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Bruton’s Tyrosine Kinase Inhibition Promotes Myelin Repair

Brain Plasticity ◽

10.3233/bpl-200100 ◽

2020 ◽

Vol 5 (2) ◽

pp. 123-133

Author(s):

Elodie Martin ◽

Marie-Stéphane Aigrot ◽

Roland Grenningloh ◽

Bruno Stankoff ◽

Catherine Lubetzki ◽

...

Keyword(s):

Tyrosine Kinase ◽

Ex Vivo ◽

Fold Increase ◽

Experimental Models ◽

Bruton’S Tyrosine Kinase ◽

Bruton's Tyrosine Kinase ◽

Myelin Repair ◽

Before And After

Background: Microglia are the resident macrophages of the central nervous system (CNS). In multiple sclerosis (MS) and related experimental models, microglia have either a pro-inflammatory or a pro-regenerative/pro-remyelinating function. Inhibition of Bruton’s tyrosine kinase (BTK), a member of the Tec family of kinases, has been shown to block differentiation of pro-inflammatory macrophages in response to granulocyte–macrophage colony-stimulating factor in vitro. However, the role of BTK in the CNS is unknown. Methods: Our aim was to investigate the effect of BTK inhibition on myelin repair in ex vivo and in vivo experimental models of demyelination and remyelination. The remyelination effect of a BTK inhibitor (BTKi; BTKi-1) was then investigated in LPC-induced demyelinated cerebellar organotypic slice cultures and metronidazole-induced demyelinated Xenopus MBP-GFP-NTR transgenic tadpoles. Results: Cellular detection of BTK and its activated form BTK-phospho-Y223 (p-BTK) was determined by immunohistochemistry in organotypic cerebellar slice cultures, before and after lysophosphatidylcholine (LPC)-induced demyelination. A low BTK signal detected by immunolabeling under normal conditions in cerebellar slices was in sharp contrast to an 8.5-fold increase in the number of BTK-positive cells observed in LPC-demyelinated slice cultures. Under both conditions, approximately 75% of cells expressing BTK and p-BTK were microglia and 25% were astrocytes. Compared with spontaneous recovery, treatment of demyelinated slice cultures and MTZ-demyelinated transgenic tadpoles with BTKi resulted in at least a 1.7-fold improvement of remyelination. Conclusion: Our data demonstrate that BTK inhibition is a promising therapeutic strategy for myelin repair.

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Dexamethasone PLGA Microspheres for Sub-Tenon Administration: Influence of Sterilization and Tolerance Studies

Pharmaceutics ◽

10.3390/pharmaceutics13020228 ◽

2021 ◽

Vol 13 (2) ◽

pp. 228

Author(s):

Deyanira Barbosa-Alfaro ◽

Vanessa Andrés-Guerrero ◽

Ivan Fernandez-Bueno ◽

María Teresa García-Gutiérrez ◽

Esther Gil-Alegre ◽

...

Keyword(s):

Particle Size ◽

In Vitro Release ◽

Intravitreal Injections ◽

Plga Microspheres ◽

Promising Alternative ◽

Before And After ◽

Evaporation Technique ◽

Release Profiles

Many diseases affecting the posterior segment of the eye require repeated intravitreal injections with corticosteroids in chronic treatments. The periocular administration is a less invasive route attracting considerable attention for long-term therapies. In the present work, dexamethasone-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (Dx-MS) were prepared using the oil-in-water (O/W) emulsion solvent evaporation technique. MS were characterized in terms of mean particle size and particle size distribution, external morphology, polymer integrity, drug content, and in vitro release profiles. MS were sterilized by gamma irradiation (25 kGy), and dexamethasone release profiles from sterilized and non-sterilized microspheres were compared by means of the similarity factor (f2). The mechanism of drug release before and after irradiation exposure of Dx-MS was identified using appropriate mathematical models. Dexamethasone release was sustained in vitro for 9 weeks. The evaluation of the in vivo tolerance was carried out in rabbit eyes, which received a sub-Tenon injection of 5 mg of sterilized Dx-MS (20–53 µm size containing 165.6 ± 3.6 µg Dx/mg MS) equivalent to 828 µg of Dx. No detectable increase in intraocular pressure was reported, and clinical and histological analysis of the ocular tissues showed no adverse events up to 6 weeks after the administration. According to the data presented in this work, the sub-Tenon administration of Dx-MS could be a promising alternative to successive intravitreal injections for the treatment of chronic diseases of the back of the eye.

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Modulation of arabinosylcytosine metabolism by arabinosyl-2- fluoroadenine in lymphocytes from patients with chronic lymphocytic leukemia: implications for combination therapy

Blood ◽

10.1182/blood.v74.6.2070.bloodjournal7462070 ◽

1989 ◽

Vol 74 (6) ◽

pp. 2070-2075 ◽

Cited By ~ 1

Author(s):

V Gandhi ◽

B Nowak ◽

MJ Keating ◽

W Plunkett

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Ex Vivo ◽

K562 Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Median Increase ◽

Cellular Concentration ◽

Before And After

Our previous studies indicated that K562 cells loaded with arabinosyl-2- fluoroadenine 5′-triphosphate (F-ara-ATP) accumulated arabinosylcytosine 5′-triphosphate (ara-CTP) at a threefold higher rate compared to the control cells. In the present study lymphocytes were obtained from patients with chronic lymphocytic leukemia before and after F-ara-A monophosphate therapy. The rate of ara-CTP accumulation after in vitro ara-C incubation was compared in lymphocytes obtained prior to therapy without any other manipulation, after ex vivo F-ara- ATP (100 mumol/L) treatment, and after in vivo F-ara-A monophosphate therapy. Lymphocytes showed a 2.2-fold (n = 23) and 1.7-fold (n = 23) median increase in the cellular concentration of ara-CTP after an ex vivo incubation with 100 mumol/L F-ara-A and 20 to 24 hours after the first dose (25 or 30 mg/m2) of F-ara-A monophosphate in vivo treatment, respectively. Although the rates of F-ara-ATP and ara-CTP accumulation varied among patients, a relationship was observed in individuals between the cellular concentration of F-ara-ATP at the beginning of the ara-C incubation and ara-CTP accumulation. These studies strongly suggest that a protocol designed to administer F-ara-A monophosphate prior to ara-C infusion will augment ara-CTP accumulation by leukemia cells.

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EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619

10.1055/s-0038-1643854 ◽

1987 ◽

Author(s):

M Hatmi ◽

A Del Maschio ◽

J Lefort ◽

G De Gaetano ◽

B B Varqaftiq ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Activation ◽

Ex Vivo ◽

Oral Treatment ◽

Human Platelets ◽

Cyclooxygenase Inhibitors ◽

Before And After ◽

A Site

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

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