Optimisation of cell and ex vivo culture conditions to study vascular calcification

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.

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Age-specific different immune responses due to capabilities of secreting primal immunoglobulins responding to unexperienced antigens

10.21203/rs.3.rs-841907/v1 ◽

2021 ◽

Author(s):

JANGHO LEE ◽

Kyoungshik Cho ◽

Hyejin Kook ◽

Suman Kang ◽

Yunsung Lee ◽

...

Keyword(s):

Stem Cells ◽

Immune System ◽

Defense Mechanism ◽

Ex Vivo ◽

Binding Capacity ◽

Culture Conditions ◽

Fetal Stem Cells ◽

Self Defense ◽

Wide Range ◽

Ex Vivo Culture

Abstract Among numerous studies on COVID-19, we noted that the infection and mortality rates of SARS-CoV-2 increased with age and that fetuses known to be particularly susceptible to infection were better protected despite various mutations. Hence, we established the hypothesis that a new immune system exists that forms before birth and decreases with aging. To prove this, we analyzed the components from early pregnancy fetal stem cells cultivated in various ex-vivo culture conditions simulating the environment during pregnancy. Resultingly, we confirmed that IgM, a natural antibody produced only in early B-1 cells, immunoglobulins including IgG3, which has a wide range of antigen-binding capacity and affinity, complement proteins, and antiviral proteins are induced. Our results suggest that fetal stem cells can form an independent immune system responding to unlearned antigens as a self-defense mechanism before establishing mature immune systems. Moreover, we propose the possibility of new solutions to cope with various infectious diseases based on the factors therein.

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Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

Blood ◽

10.1182/blood.v87.11.4589.bloodjournal87114589 ◽

1996 ◽

Vol 87 (11) ◽

pp. 4589-4595 ◽

Cited By ~ 65

Author(s):

TL Holyoake ◽

MG Freshney ◽

L McNair ◽

AN Parker ◽

PJ McKay ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Factor ◽

Ex Vivo ◽

Culture Conditions ◽

Ex Vivo Expansion ◽

Short Term ◽

Ex Vivo Culture ◽

Interleukin 11 ◽

Transplantation Model

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL- 11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.

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

Journal of Experimental Medicine ◽

10.1084/jem.186.4.619 ◽

1997 ◽

Vol 186 (4) ◽

pp. 619-624 ◽

Cited By ~ 318

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.

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Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Blood ◽

10.1182/blood.v104.11.4954.4954 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4954-4954

Author(s):

Ronald L. Brown ◽

J. Zhang ◽

L. Qiu ◽

A. Nett ◽

G. Almeida-Porada ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Culture Conditions ◽

Cell Populations ◽

Multilineage Differentiation ◽

Cd34 Cells ◽

Ex Vivo Culture ◽

Cytokine Combination ◽

Cells Cultured

Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.

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SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application

Blood ◽

10.1182/blood-2018-99-120262 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5803-5803

Author(s):

Abisha Crystal C ◽

Saravanabhavan Thangavel ◽

Shaji Ramachandran Velayudhan ◽

Alok Srivastava ◽

Aneesha Nath ◽

...

Keyword(s):

Stem Cells ◽

Cell Migration ◽

Hematopoietic Stem Cells ◽

Small Molecules ◽

Gene Editing ◽

Ex Vivo ◽

Culture Conditions ◽

Hematopoietic Stem ◽

Colony Forming Cell ◽

Ex Vivo Culture

Abstract Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process. The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+). Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. Disclosures No relevant conflicts of interest to declare.

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Forward RNAi screens in primary human hematopoietic stem/progenitor cells

Blood ◽

10.1182/blood-2008-10-176396 ◽

2009 ◽

Vol 113 (16) ◽

pp. 3690-3695 ◽

Cited By ~ 25

Author(s):

Nicole Ali ◽

Christine Karlsson ◽

Marie Aspling ◽

Guang Hu ◽

Nir Hacohen ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Ex Vivo ◽

Screening Strategy ◽

Culture Conditions ◽

Specific Gene ◽

Self Renewal ◽

Hematopoietic Stem ◽

Shrna Library ◽

Ex Vivo Culture

Abstract The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.

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Capacity for stochastic self-renewal and differentiation in mammalian spermatogonial stem cells

The Journal of Cell Biology ◽

10.1083/jcb.200907047 ◽

2009 ◽

Vol 187 (4) ◽

pp. 513-524 ◽

Cited By ~ 25

Author(s):

Zhuoru Wu ◽

Katherine Luby-Phelps ◽

Abhijit Bugde ◽

Laura A. Molyneux ◽

Bray Denard ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Cell Types ◽

Spermatogonial Stem Cells ◽

Culture Conditions ◽

Basic Question ◽

Self Renewal ◽

A Cell ◽

Fate Decision ◽

Ex Vivo Culture

Mammalian spermatogenesis is initiated and sustained by spermatogonial stem cells (SSCs) through self-renewal and differentiation. The basic question of whether SSCs have the potential to specify self-renewal and differentiation in a cell-autonomous manner has yet to be addressed. Here, we show that rat SSCs in ex vivo culture conditions consistently give rise to two distinct types of progeny: new SSCs and differentiating germ cells, even when they have been exposed to virtually identical microenvironments. Quantitative experimental measurements and mathematical modeling indicates that fate decision is stochastic, with constant probability. These results reveal an unexpected ability in a mammalian SSC to specify both self-renewal and differentiation through a self-directed mechanism, and further suggest that this mechanism operates according to stochastic principles. These findings provide an experimental basis for autonomous and stochastic fate choice as an alternative strategy for SSC fate bifurcation, which may also be relevant to other stem cell types.

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Temperature Controlled and Monitored Ex Vivo Lung Perfusion System for Research and Training Purposes

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2019-0074 ◽

2019 ◽

Vol 5 (1) ◽

pp. 293-295

Author(s):

Christina Pongratz ◽

Jens Ziegle ◽

Axel Boese ◽

Michael Friebe ◽

Helena Linge ◽

...

Keyword(s):

Storage Temperature ◽

Ex Vivo ◽

Culture Conditions ◽

Lung Perfusion ◽

Tissue Storage ◽

Ex Vivo Lung Perfusion ◽

Device Development ◽

Preservation Method ◽

Normothermic Perfusion ◽

Short Period

AbstractEx vivo lung perfusion (EVLP) is a preservation method for donor lungs, which keep lungs viable in a physiological environment outside of a body for a short period of time. EVLP is established clinically for lung transplantation. Experimental applications for EVLP are e.g. lung cancer research or medical device development and testing. For preservation, a lung is ventilated artificially in an organ chamber and perfused antegrade through the pulmonary artery. Here we introduce a thermoregulation system for an experimental EVLP system to be used for translational research approaches as well as for training medical staff. To implement physiological culture conditions that are a prerequisite for lung preservation and tissue homeostasis, a thermoregulation is needed to rewarm the explanted lung tissue (storage temperature 4°C). Technically, the EVLP system must be thermally insulated, so loss of caloric is avoided. For monitoring, temperature sensors are integrated within the lung, in the organ chamber and in the afferent perfusate tube, whereby the measured values determine the thermoregulation. Initial tests using thermal packs (cooled to 4-6°C) placed on a heating mat, as a part of the perfusion circuit, showed that the perfusate temperature falls to 34°C, but restores after approximately 60 minutes (36.5°C), whereby the thermal pack is warmed. With this setup longer perfusion times should be obtained rather than without thermoregulation due to normothermic perfusion of the lung.

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