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1.
Cytotherapy ; 21(6): 631-642, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30975604

RESUMEN

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.


Asunto(s)
Materiales Biocompatibles/química , Regeneración Ósea/fisiología , Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Animales , Reactores Biológicos , Recuento de Células , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Citocinas/metabolismo , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/fisiología , Poliésteres/química , Ratas Desnudas , Cráneo
2.
Cytotherapy ; 19(3): 419-432, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28017598

RESUMEN

Large numbers of human mesenchymal stromal cells (MSCs) used for a variety of applications in tissue engineering and cell therapy can be generated by scalable expansion in a bioreactor using microcarriers (MCs) systems. However, the enzymatic digestion process needed to detach cells from the growth surface can affect cell viability and potentially the potency and differentiation efficiency. Thus, the main aim of our study was to develop biocompatible and biodegradable MCs that can support high MSC yields while maintaining their differentiation capability and potency. After cell expansion, the cells that covered MCs can be directly implanted in vivo without the need for cell harvesting or use of scaffold. Poly-ε-caprolactone (PCL) is known as a biocompatible and biodegradable material. However, it cannot be used for generation of MCs because its high density (1.14 g/cm3) would exclude its applicability for suspension MCs in stirred reactors. In this article, we describe expansion and potency of MSCs propagated on low-density (1.06 g/cm3) porous PCL MCs coated with extracellular matrices (LPCLs) in suspended stirred reactors. Using these LPCLs, cell yields of about 4 × 104 cells/cm2 and 7- to 10-fold increases were obtained using four different MSC lines (bone marrow, cord blood, fetal and Wharton's jelly). These yields were comparable with those obtained using non-degradable MCs (Cytodex 3) and higher than two-dimensional monolayer (MNL) cultures. A fed-batch process, which demonstrated faster cell expansion (4.5 × 104 cells/cm2 in 5 days as compared with 7 days in batch culture) and about 70% reduction in growth media usage, was developed and scaled up from 100-mL spinner flask to 1-L controlled bioreactor. Surface marker expression, trilineage differentiation and clonogenic potential of the MSCs expanded on LPCL were not affected. Cytokine secretion kinetics, which occurred mostly during late logarithmic phase, was usually comparable with that obtained in Cytodex 3 cultures and higher than MNL cultures. In conclusion, biodegradable LPCL can be used to efficiently expand a variety of MSC lines in stirred scalable reactors in a cost-effective manner while maintaining surface markers expression, differentiation capability and high levels of cytokine secretion. This study is the first step in testing these cell-biodegradable porous MC aggregates for tissue engineering and cell therapy, such as bone and cartilage regeneration, or wound healing.


Asunto(s)
Implantes Absorbibles , Técnicas de Cultivo Celular por Lotes/métodos , Proliferación Celular , Citocinas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Poliésteres/química , Andamios del Tejido/química , Reactores Biológicos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Medios de Cultivo/metabolismo , Dextranos/química , Humanos , Ensayo de Materiales , Microtecnología/instrumentación , Ingeniería de Tejidos/métodos
3.
Langmuir ; 33(12): 3068-3079, 2017 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-28221044

RESUMEN

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm3. Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm3, requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm3. The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 µm and densities ranging from 1.05-1.11 g/cm3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm3, achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.


Asunto(s)
Células Madre Mesenquimatosas/citología , Poliésteres/química , Supervivencia Celular , Humanos , Cloruro de Metileno/química , Microesferas , Estructura Molecular , Tamaño de la Partícula , Porosidad , Propiedades de Superficie
4.
Biochem Biophys Res Commun ; 473(3): 769-73, 2016 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-26385177

RESUMEN

Mesenchymal stromal cells (MSCs) are being investigated for a variety of therapeutic indications. However, current 2D planar technology cannot meet the anticipated demand and a shift to serum-free microcarrier cultures is needed in order to meet the quality and quantity of cells required. Here we summarize several recent attempts to grow cells in such conditions, and identify several variables that affect cell expansion, including tissue source, serum-free medium formulation, microcarrier type and matrix, and agitation regime (continuous versus intermittent). Optimization of these culture conditions will be necessary to ensure success in bioreactor-scale production of MSCs for cell therapies.


Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Mesenquimatosas/citología , Animales , Reactores Biológicos , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Medios de Cultivo/metabolismo , Matriz Extracelular/metabolismo , Humanos , Fenotipo , Resistencia al Corte
5.
Biochem Biophys Res Commun ; 473(3): 764-8, 2016 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-26385176

RESUMEN

Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles of such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Neuronas/metabolismo , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Humanos , Miocitos Cardíacos/citología , Células Madre/citología
6.
Cytotherapy ; 18(6): 740-53, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27173750

RESUMEN

BACKGROUND AIMS: Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. METHODS: heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100ß, MMP13 and ALPL, were investigated and compared in both conditions. RESULTS: BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100ß) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. CONCLUSIONS: This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.


Asunto(s)
Cartílago/metabolismo , Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Condrogénesis/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Fosfatasa Alcalina/biosíntesis , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/citología , Colágeno/metabolismo , ADN/análisis , ADN/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Metaloproteinasa 13 de la Matriz/biosíntesis , Subunidad beta de la Proteína de Unión al Calcio S100/biosíntesis , Factor de Transcripción SOX9/biosíntesis , Trasplante Homólogo
7.
Cytotherapy ; 18(10): 1332-44, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27503763

RESUMEN

BACKGROUND AIMS: Human mesenchymal stromal cells or marrow stromal cells (MSCs) are of great interest for bone healing due to their multi-potency and trophic effects. However, traditional MSC expansion methods using 2-dimensional monolayer (MNL) flasks or cell stacks are limited by labor-intensive handling, lack of scalability, the need for enzymatic cell harvesting and the need for attachment to a scaffold before in vivo delivery. Here, we present a biodegradable microcarrier and MSC bioprocessing system that may overcome the abovementioned challenges. METHODS: We cultured human early MSCs (heMSCs) on biodegradable polycaprolactone microcarriers (PCL MCs) coated with extracellular matrix (ECM) and evaluated the in vitro osteogenic differentiation and in vivo bone formation capacity of ECM-coated PCL MC-bound heMSCs compared with conventional MNL-cultured cells. RESULTS: We found that heMSCs proliferate well on PCL MCs coated with a fibronectin, poly-l-lysine, and fibronectin (FN+PLL+FN) coating (cPCL MCs). During in vitro osteogenic induction, heMSCs cultured on cPCL MCs displayed a 68% increase in specific calcium deposition compared with cultures on MNL. In a mouse ectopic mineralization model, bone mass was equivalent for MNL-expanded and cPCL MC-bound heMSC implants but higher in both cases when compared with cell-free cPCL MC implants at 16 weeks post-implantation. In summary, compared with MNL cultures, biodegradable MC MSC cultures provide the benefits of large-scale expansion of cells and can be delivered in vivo, thereby eliminating the need for cell harvesting and use of scaffolds for cell delivery. These results highlight the promise of delivering heMSCs cultured on cPCL MCs for bone applications.


Asunto(s)
Implantes Absorbibles , Proliferación Celular , Matriz Extracelular/química , Células Madre Mesenquimatosas/fisiología , Miniaturización , Osteogénesis/fisiología , Poliésteres/química , Andamios del Tejido/química , Animales , Regeneración Ósea/efectos de los fármacos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Microtecnología , Miniaturización/instrumentación , Miniaturización/métodos , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología
8.
J Mol Cell Cardiol ; 80: 56-70, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25528965

RESUMEN

Differentiation of human pluripotent stem cells as embryoid bodies (EBs) has been achieved previously with p38alfa MAPK inhibitors such as SB203580 with moderate efficiency of 10-15%. We synthesized and screened 42 compounds that are 2,4,5-trisubstituted azole analogues of SB203580 for efficient cardiomyocyte differentiation. Our screen identified novel compounds that have similar cardiac differentiation activity as SB203580. However, the cardiac differentiation did not correlate with p38alfa MAPK inhibition, indicating an alternative mechanism in cardiac differentiation. Upon profiling several 2,4,5-trisubstituted azole compounds against a panel of 97 kinases we identified several off targets, among them casein kinases 1 (CK1). The cardiomyogenic activities of SB203580 and its analogues showed a correlation with post mesoderm Wnt/beta-catenin pathway inhibition of CK1 epsilon and delta. These findings united the mechanism of 2,4,5-trisubstituted azole with the current theory of Wnt/beta-catenin regulated pathway of cardiac differentiation. Consequently an efficient cardiomyocyte protocol was developed with Wnt activator CHIR99021 and 2,4,5-trisubstituted azoles to give high yields of 50-70% cardiomyocytes and a 2-fold increase in growth.


Asunto(s)
Quinasa de la Caseína I/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Piridinas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Línea Celular , Diseño de Fármacos , Humanos , Imidazoles/síntesis química , Mesodermo/citología , Mesodermo/efectos de los fármacos , Ratones , Organogénesis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/síntesis química
9.
BMC Biotechnol ; 15: 102, 2015 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-26520400

RESUMEN

BACKGROUND: Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. However, challenges to large-scale production of MSCs can hinder the translation of MSC therapies. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effective alternative to conventional methods of expansion in 2D monolayers. Furthermore, biodegradable MCs may allow for MC-bound MSC delivery without enzymatic harvest for selected applications such as bone healing. However, the effects of cell expansion on microcarriers and enzymatic cell harvest on MSC phenotype and osteogenic differential potential are not well understood. In this study, we characterized human fetal MSCs (hfMSCs) after expansion in 3D microcarrier spinner or 2D monolayer cultures. Following expansion, we compared osteogenic differentiation of cultures seeded with 3D MC-harvested, 3D MC-bound and conventional 2D monolayer (MNL)-harvested cells when cultured in osteogenic induction media on collagen-coated plates. RESULTS: Fetal MSCs expanded on both 3D agitated Microcarriers (MC) and 2D Plastic static monolayer (MNL) cultures express high levels of MSC surface markers. MC-harvested hfMSCs displayed higher expression of early osteogenic genes but slower mineralization kinetics compared to MNL-harvested MSCs during osteogenic induction. However, in the comparison between MC-bound and MC-harvested hfMSCs, osteogenic genes were upregulated and mineralization kinetics was accelerated in the former condition. Importantly, 3D MC-bound hfMSCs expressed higher levels of osteogenic genes and displayed either higher or equivalent levels of mineralization, depending on the cell line, compared to the classical monolayer cultures use in the literature (MNL-harvested hfMSCs). CONCLUSION: Beyond the processing and scalability advantages of the microcarrier culture, hfMSCs attached to MCs undergo robust osteogenic differentiation and mineralization compared to enzymatically harvested cells. Thus biodegradable/biocompatible MCs which can potentially be used for cell expansion as well as a scaffold for direct in vivo delivery of cells may have advantages over the current methods of monolayer-expansion and delivery post-harvest for bone regeneration applications.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/fisiología , Osteogénesis/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Ensayo de Inmunoadsorción Enzimática , Feto/citología , Feto/fisiología , Citometría de Flujo , Humanos , Técnicas In Vitro
10.
Cytotherapy ; 17(2): 163-73, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25304664

RESUMEN

BACKGROUND AIMS: Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. METHODS: MSCs were expanded in stirred cultures on Cytodex 3 microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. RESULTS: Minimum essential medium-α-based medium supported faster MSC growth on microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 10(6) cells/mL with confluent cell concentration of 4.7 × 10(4)cells/cm(2) was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10(6)cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10(6)cells. CONCLUSIONS: We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.


Asunto(s)
Reactores Biológicos , Dextranos , Células Madre Mesenquimatosas/citología , Microesferas , Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos , Medios de Cultivo , Glucosa , Humanos , Trasplante de Células Madre Mesenquimatosas
11.
Cytotherapy ; 17(8): 1152-65, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26139547

RESUMEN

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. METHODS: We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. RESULTS: We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. CONCLUSIONS: Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line.


Asunto(s)
Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Medio de Cultivo Libre de Suero , Células Madre Mesenquimatosas/citología , Línea Celular , Proliferación Celular , Humanos
12.
Biomed Microdevices ; 17(6): 105, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26458560

RESUMEN

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres, which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution, as well as the resulting PCL microsphere size, are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets, within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c, the microfluidic device is operated in dripping mode, which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity, resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins, the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells.


Asunto(s)
Células Madre Embrionarias Humanas/citología , Microesferas , Poliésteres/química , Adhesión Celular , Proliferación Celular , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Proteínas de la Matriz Extracelular/química , Humanos , Peso Molecular , Tamaño de la Partícula , Viscosidad
13.
Immunol Rev ; 239(1): 221-36, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21198675

RESUMEN

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.


Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Animales , Carbunco/terapia , Anticuerpos Antibacterianos/inmunología , Bacillus anthracis/inmunología , Bacillus anthracis/patogenicidad , Guerra Biológica , Modelos Animales de Enfermedad , Cobayas , Humanos , Ratones , Conejos , Ratas , Esporas Bacterianas/inmunología , Esporas Bacterianas/patogenicidad , Vacunas de ADN
15.
Bioorg Med Chem Lett ; 23(11): 3300-3, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23602399

RESUMEN

The p38α mitogen-activated protein kinase (MAPK) inhibitor SB203580 had been reported to enhance the cardiomyogenesis of human embryonic stem cells (hESCs). To investigate if tri-substituted imidazole analogues of SB203580 are equally effective inducers for cardiomyogenesis of hESCs, and if there is a correlation between p38α MAPK inhibition and cardiomyogenesis, we designed and synthesized a series of novel tri-substituted imidazoles with a range of p38α MAPK inhibitory activities. Our studies demonstrated that suitably designed analogues of SB203580 can also be inducers of cardiomyogenesis in hESCs and that cell growth is affected by changes in the imidazole structures.


Asunto(s)
Células Madre Embrionarias/citología , Imidazoles/química , Piridinas/química , Diferenciación Celular/efectos de los fármacos , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/citología , Unión Proteica , Piridinas/metabolismo
16.
Methods Mol Biol ; 2436: 67-81, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34519977

RESUMEN

Human-induced pluripotent stem cells are known for their high proliferation capacity as well as their ability to differentiate to different lineages (Ban et al., Theranostics 7(7):2067-2077, 2017; Chen et al., Stem Cell Res 15(2):365-375, 2015; Serra et al., Trends Biotechnol 30(6):350-359, 2012). For stem-cell-derived cardiomyocytes to evolve into a scalable therapeutic source, a large quantity of highly pure cardiomyocytes is needed. Thus, lies the challenge of defining an efficient cardiomyocyte differentiation process. This chapter describes a method to evaluate multiple human-induced pluripotent stem cell lines for their cardiac differentiation potentials before evaluating their integrated proliferation and differentiation abilities in microcarrier cultures in a spinner culture format.


Asunto(s)
Células Madre Pluripotentes Inducidas , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Humanos , Pruebas Inmunológicas , Miocitos Cardíacos
17.
Cell Prolif ; : e13256, 2022 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-36574589

RESUMEN

OBJECTIVES: Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. MATERIALS AND METHODS: Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. RESULTS: Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (ß-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. CONCLUSIONS: Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

18.
Cell Prolif ; 55(8): e13218, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-35289971

RESUMEN

OBJECTIVES: Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. MATERIALS AND METHODS: We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. RESULTS: Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. CONCLUSIONS: This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.


Asunto(s)
Células Madre Pluripotentes Inducidas , Reactores Biológicos , Diferenciación Celular , Células Clonales , Eritrocitos , Eritropoyesis , Humanos , Perfusión
19.
Stem Cell Res ; 53: 102272, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33676128

RESUMEN

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.


Asunto(s)
Células Madre Mesenquimatosas , MicroARNs , Reactores Biológicos , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/genética , Humanos , MicroARNs/genética
20.
Stem Cell Reports ; 16(1): 182-197, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33306988

RESUMEN

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Eritrocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular , Células Cultivadas , Eritrocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma
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