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1.
Biotechnol J ; 13(2)2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29178199

RESUMO

The adult bone marrow (BM) niche is a complex entity where a homeostatic hematopoietic system is maintained through a dynamic crosstalk between different cellular and non-cellular players. Signaling mechanisms triggered by cell-cell, cell-extracellular matrix (ECM), cell-cytokine interactions, and local microenvironment parameters are involved in controlling quiescence, self-renewal, differentiation, and migration of hematopoietic stem/progenitor cells (HSPC). A promising strategy to more efficiently expand HSPC numbers and tune their properties ex vivo is to mimic the hematopoietic niche through integration of adjuvant stromal cells, soluble cues, and/or biomaterial-based approaches in HSPC culture systems. Particularly, mesenchymal stem/stromal cells (MSC), through their paracrine activity or direct contact with HSPC, are thought to be a relevant niche player, positioning HSPC-MSC co-culture as a valuable platform to support the ex vivo expansion of hematopoietic progenitors. To improve the clinical outcome of hematopoietic cell transplantation (HCT), namely when the available HSPC are present in a limited number such is the case of HSPC collected from umbilical cord blood (UCB), ex vivo expansion of HSPC is required without eliminating the long-term repopulating capacity of more primitive HSC. Here, we will focus on depicting the characteristics of co-culture systems, as well as other bioengineering approaches to improve the functionality of HSPC ex vivo.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Animais , Biomimética , Reatores Biológicos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Modelos Animais
2.
J Tissue Eng Regen Med ; 11(5): 1630-1640, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27444977

RESUMO

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGROTM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Plaquetas/química , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/metabolismo
3.
J Biotechnol ; 236: 88-109, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27527397

RESUMO

Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Mesenquimais , Animais , Reatores Biológicos , Células Cultivadas , Humanos , Camundongos
4.
J Tissue Eng Regen Med ; 9(10): 1172-81, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23596131

RESUMO

Physiologically low O(2) tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0-5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O(2) tensions (3-10%). We hypothesized that O(2) concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34(+) -enriched cells was studied in co-culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O(2). 2% O(2) resulted in a significantly lower CD34(+) cell expansion (25-fold vs 60-, 64- and 92-fold at day 10 for 5%, 21%, 10% O(2), respectively). In turn, 10% O(2) promoted the highest CD34(+) CD90(+) cell expansion, reaching 22 ± 5.4- vs 5.6 ± 2.4- and 5.7 ± 2.0-fold for 2%, 5% and 21% O(2), respectively, after 14 days. Similar differentiation patterns were observed under different O(2) tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O(2). Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O(2) when compared to higher O(2) concentrations (5-21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O(2), can maximize the ex vivo expansion of UCB stem/progenitor cells in co-culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications.


Assuntos
Diferenciação Celular , Proliferação de Células , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Oxigênio , Antígenos CD34/imunologia , Técnicas de Cocultura , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas In Vitro , Antígenos Thy-1/imunologia
5.
Chem Phys Lipids ; 165(5): 577-88, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22705749

RESUMO

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (l(o)) and liquid disordered (l(d)) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase. N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label l(d) domains in yeast membranes. 4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to l(o) domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol-lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.


Assuntos
Fenômenos Biofísicos , Ergosterol/metabolismo , Corantes Fluorescentes/metabolismo , Microdomínios da Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Saccharomyces cerevisiae/citologia , Ergosterol/química , Corantes Fluorescentes/química , Fosfatidilcolinas/química , Espectrometria de Fluorescência
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