Bone marrow–on–a–chip replicates hematopoietic niche physiology in vitro

Abstract

____________________________________________________________________________The bone marrow microenvironment contains a complex set of cellular, chemical, structural and physical cues necessary to maintain the viability and function of the hematopoietic system [1][2][3][4][5] . This hematopoietic niche regulates hematopoietic stem cells (HSCs), facilitating a delicate balance between self-renewal and differentiation into progenitor cells that produce all mature blood cell types 4,5 . Engineering an artificial bone marrow that reconstitutes natural marrow structure and function, and that can be maintained in culture, could be a powerful platform to study hematopoiesis and test new therapeutics. It has proven difficult, however, to recreate the complex bone marrow microenvironment needed to support the formation and maintenance of a complete, functional hematopoietic niche in vitro [6][7][8][9] . Although various in vitro culture systems have (Revised NMETH-A19608) been developed to maintain and expand hematopoietic stem and progenitor cells 6-11 , there is currently no method to recreate or study the intact bone marrow microenvironment in vitro. Therefore, studies on hematopoiesis commonly rely on animal models to ensure the presence of an intact bone marrow microenvironment that enables normal physiologic marrow responses [12][13][14][15] . Furthermore, although it has been reported that bone marrow can be engineered in vivo [16][17][18][19] , no method exists to culture engineered bone marrow in vitro. To bridge the functional gap between in vivo and in vitro systems, we developed a method to produce a "bone marrow-on-a-chip" culture system that contains artificial bone and a living marrow, which is first generated in mice, and then explanted whole and maintained in vitro within a microfluidic device. RESULTS In vivo engineering of bone marrowTissue engineering methods have been used to induce formation of new bone with a central marrow compartment in vivo [18][19][20][21] . To explore the possibility of engineering an artificial bone marrow that can be explanted whole, we microfabricated a polydimethylsiloxane (PDMS) device with a central cylindrical cavity (1 mm high x 4 mm diameter) with openings at both ends (Fig. 1a). We filled the hollow compartment with a type I collagen gel containing bone-inducing demineralized bone powder (DBP) and bone morphogenetic proteins (BMP2 and BMP4) [20][21][22] , and implanted it subcutaneously on the back of a mouse (Supplementary Fig. 1). Our goal was to engineer bone that would fill the cylindrical space within the implanted device so that it could be easily removed whole and inserted into a microfluidic system containing a similarly shaped (Revised NMETH-A19608) chamber for in vitro culture (Fig. 1 a,b). These initial studies resulted in the creation of new bone encasing a marrow compartment that formed within the PDMS device 4 to 8 weeks after subcutaneous implantation. Histological analysis revealed that the marrow was largely inhabited by adipocytes and exhibite...

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