RESUMO
We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.
Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Dispositivos Lab-On-A-Chip , Megacariócitos/citologia , Modelos Biológicos , Antígenos CD/fisiologia , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Biomimética , Reatores Biológicos , Plaquetas/fisiologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Sangue Fetal/fisiologia , Expressão Gênica , Humanos , Megacariócitos/fisiologia , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Contagem de Plaquetas , Reologia , Estresse MecânicoRESUMO
We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.