Our team worked in conjunction with our faculty advisor Dr. Jing Pan to devise a step-by-step protocol for Wet Lab Members to create our bone-marrow organoids.
Aim 1
Aim 1 includes the culturing steps of iPSCs, with a focus on sterile lab practices and documentation. Aim 1 includes matrigel coating of 6-well plates, thawing of iPSCs, iPSC maintenance, passaging of iPSCs, as well as cryopreservation. Propagation of human iPSCs is a difficult process, as cells must be grown in specific temperature conditions and can grow on top of one another. Our goal was to generate a continuous propagation of iPSCs, and once a passaged plate showed viable, undifferentiated cell colonies at 60% confluence, Aim 2 could be initiated.
Aim 2
Aim 2 describes the conversion of undifferentiated iPSC cell colonies to a vascularized, organoid system with structural and chemical similarities to human bone marrow. Through the careful addition of growth factors and collagen, we can induce vascular sprouting that replicates the hematopoietic niche of bone marrow. Our goals are to observe a highly vascularized space with specialized sinusoidal endothelium, hematopoiesis, transmigration through sinusoids, and specialized stroma. Aim 2 is a 12-day cycle, with 3 Phases of medium containing growth factors that encourage cells to commit to vascular and hematopoietic lineages.