Effect of Long-Term 3D Spheroid Culture on WJ-MSC

The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.

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Activation of the receptor tyrosine kinase RET improves long-term hematopoietic stem cell outgrowth and potency

Blood ◽

10.1182/blood.2020006302 ◽

2020 ◽

Vol 136 (22) ◽

pp. 2535-2547 ◽

Cited By ~ 5

Author(s):

W. Grey ◽

R. Chauhan ◽

M. Piganeau ◽

H. Huerga Encabo ◽

M. Garcia-Albornoz ◽

...

Keyword(s):

Intracellular Signaling ◽

Culture Conditions ◽

Cellular Growth ◽

Great Promise ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Intracellular Signaling Pathway ◽

Wide Range

Abstract Expansion of human hematopoietic stem cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche; how to apply this process to HSC maintenance and expansion has yet to be explored. We show a role for the GFL receptor, RET, at the cell surface of HSCs in mediating sustained cellular growth, resistance to stress, and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/coreceptor complex, glial-derived neurotrophic factor and its coreceptor, exhibit improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we show that RET drives a multifaceted intracellular signaling pathway, including key signaling intermediates protein kinase B, extracellular signal-regulated kinase 1/2, NF-κB, and p53, responsible for a wide range of cellular and genetic responses that improve cell growth and survival under culture conditions.

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Structure of vegetative organs in essential oil rose under standard culture conditions and long-term conservation in vitro

Acta Horticulturae ◽

10.17660/actahortic.2021.1315.28 ◽

2021 ◽

pp. 185-190

Author(s):

I.V. Mitrofanova ◽

V.A. Brailko ◽

N.P. Lesnikova-Sedoshenko ◽

N.N. Ivanova ◽

O.V. Mitrofanova

Keyword(s):

Essential Oil ◽

Culture Conditions ◽

Vegetative Organs ◽

Standard Culture

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Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽

10.1182/blood.v88.11.4149.4149 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4149-4158 ◽

Cited By ~ 3

Author(s):

M Trevisan ◽

XQ Yan ◽

NN Iscove

Keyword(s):

Colony Formation ◽

Interleukin 1 ◽

Methyl Cellulose ◽

Culture Conditions ◽

Liquid Suspension ◽

Almost All ◽

Marrow Cells

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

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Gravity-Based Flow Efficient Perfusion Culture System for Spheroids Mimicking Liver Inflammation

Biomedicines ◽

10.3390/biomedicines9101369 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1369

Author(s):

Young-Su Kim ◽

Arun Asif ◽

Abdul Rahim Chethikkattuveli Salih ◽

Jae-Wook Lee ◽

Ki-Nam Hyun ◽

...

Keyword(s):

Culture System ◽

Disease Modeling ◽

Perfusion Culture ◽

Drug Analysis ◽

Culture Conditions ◽

Current Approach ◽

Spheroid Culture ◽

Organ Specific ◽

Static Conditions

The spheroid culture system provides an efficient method to emulate organ-specific pathophysiology, overcoming the traditional two-dimensional (2D) cell culture limitations. The intervention of microfluidics in the spheroid culture platform has the potential to enhance the capacity of in vitro microphysiological tissues for disease modeling. Conventionally, spheroid culture is carried out in static conditions, making the media nutrient-deficient around the spheroid periphery. The current approach tries to enhance the capacity of the spheroid culture platform by integrating the perfusion channel for dynamic culture conditions. A pro-inflammatory hepatic model was emulated using a coculture of HepG2 cell line, fibroblasts, and endothelial cells for validating the spheroid culture plate with a perfusable channel across the spheroid well. Enhanced proliferation and metabolic capacity of the microphysiological model were observed and further validated by metabolic assays. A comparative analysis of static and dynamic conditions validated the advantage of spheroid culture with dynamic media flow. Hepatic spheroids were found to have improved proliferation in dynamic flow conditions as compared to the static culture platform. The perfusable culture system for spheroids is more physiologically relevant as compared to the static spheroid culture system for disease and drug analysis.

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Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽

10.1182/blood.v68.6.1348.1348 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1348-1354 ◽

Cited By ~ 53

Author(s):

A Johnson ◽

K Dorshkind

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Stromal Cell ◽

Culture Conditions ◽

B Lymphopoiesis ◽

Bone Marrow Cultures ◽

Factor Secretion ◽

Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

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Performance of Polyester-Based Electrospun Scaffolds under In Vitro Hydrolytic Conditions: From Short-Term to Long-Term Applications

Nanomaterials ◽

10.3390/nano9050786 ◽

2019 ◽

Vol 9 (5) ◽

pp. 786 ◽

Cited By ~ 3

Author(s):

Oscar Gil-Castell ◽

José David Badia ◽

Jordi Bou ◽

Amparo Ribes-Greus

Keyword(s):

Molar Mass ◽

Pure Water ◽

Phosphate Buffer Solution ◽

Short Term ◽

Buffer Solution ◽

Electrospun Scaffolds ◽

The Stability ◽

Short Time

The evaluation of the performance of polyesters under in vitro physiologic conditions is essential to design scaffolds with an adequate lifespan for a given application. In this line, the degradation-durability patterns of poly(lactide-co-glycolide) (PLGA), polydioxanone (PDO), polycaprolactone (PCL) and polyhydroxybutyrate (PHB) scaffolds were monitored and compared giving, as a result, a basis for the specific design of scaffolds from short-term to long-term applications. For this purpose, they were immersed in ultra-pure water and phosphate buffer solution (PBS) at 37 °C. The scaffolds for short-time applications were PLGA and PDO, in which the molar mass diminished down to 20% in a 20–30 days lifespan. While PDO developed crystallinity that prevented the geometry of the fibres, those of PLGA coalesced and collapsed. The scaffolds for long-term applications were PCL and PHB, in which the molar mass followed a progressive decrease, reaching values of 10% for PCL and almost 50% for PHB after 650 days of immersion. This resistant pattern was mainly ascribed to the stability of the crystalline domains of the fibres, in which the diameters remained almost unaffected. From the perspective of an adequate balance between the durability and degradation, this study may serve technologists as a reference point to design polyester-based scaffolds for biomedical applications.

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Acellular Bone Colonization and Aggregate Culture Conditions Diversely Influence Murine Periosteum Mesenchymal Stem Cell Differentiation Potential in Long-Term In Vitro Osteoinductive Conditions

Tissue Engineering Part A ◽

10.1089/ten.tea.2011.0411 ◽

2012 ◽

Vol 18 (13-14) ◽

pp. 1509-1519 ◽

Cited By ~ 3

Author(s):

Federico Ferro ◽

Renza Spelat ◽

Federica D'Aurizio ◽

Giuseppe Falini ◽

Ilaria De Pol ◽

...

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Mesenchymal Stem Cell ◽

Stem Cell Differentiation ◽

Culture Conditions ◽

Differentiation Potential ◽

Mesenchymal Stem Cell Differentiation ◽

Aggregate Culture

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Suboptimal in vitro culture conditions: an epigenetic origin of long-term health effects

Molecular Reproduction and Development ◽

10.1002/mrd.20746 ◽

2007 ◽

Vol 74 (9) ◽

pp. 1149-1156 ◽

Cited By ~ 59

Author(s):

Raúl Fernández-Gonzalez ◽

Miguel Angel Ramirez ◽

Ainhoa Bilbao ◽

Fernando Rodríguez De Fonseca ◽

Alfonso Gutiérrez-Adán

Keyword(s):

In Vitro Culture ◽

Health Effects ◽

Culture Conditions

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Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽

10.1182/blood.v120.21.3546.3546 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3546-3546

Author(s):

Sawa Ito ◽

A. John Barrett ◽

Andre Larochelle ◽

Nancy F. Hensel ◽

Keyvan Keyvanfar ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Culture Conditions ◽

Myelogenous Leukemia ◽

Cell Cycle Analysis ◽

Remission Induction ◽

Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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