Silk4B’s silk-based 3D bone marrow models allow differentiation into enucleated erythrocytes, or red blood cells (RBCs), that resemble peripheral RBCs found in the human body.

The differentiation process mimics in vivo steps with sequential differentiation into erythroblastic islands (EBIs). The morphology of erythroblastic islands in our models resembles that of in vivo EBIs. The EBIs in our models contain a gradient of less mature CD71+ CD235- cells to more mature CD71low CD235+ cells. Transmission electron microscopy clearly identifies cells from progenitor ProE cells to Rtics. Eventually, enucleated biconcave RBCs form at the periphery of EBIs. Collected RBCs are primarily enucleated and are similar to peripheral RBCs, they show very few residual mitochondria and glycogen granules. 

RBCs can easily be isolated and used in downstream assays. RBCs collection is easy from both silk hydrogel and silk scaffold models. Scaffold models allow for perfusion of the model with platelets released into the perfuate mimicking the RBC release into the blood. The release of RBCs into the perfuate allows their easy collection. 

When compared to 2D liquid culture models, RBC differentiation in our model results in differentiation of significantly more mature cells that are enucleated. This is supported by comparative transcriptomic analyses which show that unlike 2D liquid culture, our 3D bone marrow models support autophagy processes which promote organelle clearance and membrane remodelling. Importantly, in 2D culture cells experience significant stress that causes increased gamma-globin production; while in our 3D models cells show less gamma-globin production due to lower stress.