RESUMEN
Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.
Asunto(s)
Materiales Biomiméticos/química , Dimetilpolisiloxanos/química , Células Madre Hematopoyéticas/metabolismo , Andamios del Tejido/química , Transcriptoma , Adulto , Materiales Biomiméticos/efectos adversos , Proliferación Celular , Células Cultivadas , Dimetilpolisiloxanos/efectos adversos , Femenino , Factores de Transcripción Forkhead/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Andamios del Tejido/efectos adversosRESUMEN
By the use of a MatriGrid® we have established a three-dimensional high density cell culture. The MatriGrid® is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300 µm diameter and 207 µm deep) the cells can growth three-dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three-dimensional static and three-dimensional actively perfused. The results show that the cells in a three-dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer culture.