Micromarrows—Three-Dimensional Coculture of Hematopoietic Stem Cells and Mesenchymal Stromal Cells

Cook, Matthew; Futrega, Kathryn; Osiecki, Michael; Kabiri, Mahboubeh; Kul, Betul; Rice, Alison; Atkinson, Kerry; Brooke, Gary; Doran, Michael

doi:10.1089/ten.tec.2011.0159

Cited by 59 publications

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“…3 a,b). Thus, the long-term HSCs, which are the only cells capable of long-term self-renewal and multi-lineage potential, appear to be differentiating into more specialized progenitor cells in the static stromasupported cultures system, as previously reported [6][7][8][9] . In contrast, the number and distribution of HSCs and hematopoietic progenitor cells in the eBM cultured for up to 7 days on-chip were maintained in similar proportions to freshly harvested bone marrow (Fig.…”

Section: In Vitro Culture Of Engineered Bone Marrowsupporting

confidence: 71%

“…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.…”

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confidence: 99%

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

et al. 2014

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____________________________________________________________________________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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Section: In Vitro Culture Of Engineered Bone Marrowsupporting

confidence: 71%

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confidence: 99%

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

et al. 2014

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“…Furthermore, studies have indicated that HSC--supportive properties of both human [314,330] and murine [329,341] MSC are maintained when cultured as spheroids. de Barros et al developed a 3D MSC spheroid culture system with the capacity for HSPC lodgment and proliferation [331].…”

Section: Introductionmentioning

confidence: 99%

“…CB--derived CD34 + progenitor cells were reported to attach and migrate into the MSC spheroids. In previous work, we described a high--throughput 3D spheroid co--culture system for expanding murine HSPC in the presence of MSC [341]. Spheroids were manufactured in microwell plates (AggreWell, StemCell Technologies), yielding thousands of 3D spheroids [341].…”

Section: Introductionmentioning

confidence: 99%

“…In previous work, we described a high--throughput 3D spheroid co--culture system for expanding murine HSPC in the presence of MSC [341]. Spheroids were manufactured in microwell plates (AggreWell, StemCell Technologies), yielding thousands of 3D spheroids [341]. Murine 3D co--cultures enabled an approximate 2--fold increase in the yield of HSPC following 7--days of culture, relative to 2D controls.…”

Section: Introductionmentioning

confidence: 99%

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Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

Futrega¹

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KeywordsAIM 2: Utilization of the high throughput microwell platform to manufacture HSPC/MSC spheroids to improve the efficacy of direct BM transplantation.AIM 3: Development of an improved microwell platform that retains spheroids within discrete microwells throughout culture. AIM 4:Characterization of HSPC surface marker change in response to microwell material. DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHEROID CULTURES iiiChapter 2 describes the development of a high throughput HSPC/MSC 3D spheroid co--culture embodied in AIM 1. I developed a high throughput polydimethylsiloxane (PDMS) microwell platform that enabled the manufacture of thousands of uniform 3D multicellular co--culture spheroids. Spheroids were assembled such that each contained 25--to--400 BM--derived MSC and 10 CB--derived CD34 + cells (a cell population enriched for HSPC). The outcomes from this Chapter guided the design of the subsequent three chapters. The specific outcomes and subsequent study results were as follows:Chapter 3: As described in Chapter 2, only modest improvements in expansion could be achieved by co--culturing HSPC in 3D MSC spheroids using a microwell platform. We reasoned that the 3D spheroids could, however, function as a biologically active anchor to improve direct BM transplantation outcomes. In this Chapter, we contrasted transplantation methods, including the use of MSC suspensions and 3D multicellular spheroids to modulate human CB--derived CD34 + cell retention within the BM cavity of NOD/SCID gamma (NSG) mice. Analysis revealed that the BM of injected femurs in some animals was saturated with human cells, while distal haematopoietic tissue engraftment was poor. These data show, for the first time, that retention of human haematopoietic cells within the BM enhances local engraftment at the expense of systemic engraftment.Chapter 4: As identified in Chapter 2, improvements in 3D spheroid co--cultures will require that the microwell platform is compatible with repeated medium exchange or dilution feeding. Multiple partial or complete medium exchanges can displace spheroids from discrete microwells, and this can result in either the loss of spheroids from culture, or spheroid amalgamation when displaced microtissues fall into common microwells. In this Chapter, I describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells. We called this platform the microwell--mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells.Chapter 5: As identified in Chapter 2, the presence of PDMS, the material used to fabricate the microwell platform, altered CD38 surface expression on CB--derived CD34 + cells. Previous reports have shown that inhibition of retinoic acid signaling down--regulated CD38 iv DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHE...

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Stem‐Cell Niche Based Comparative Analysis of Chemical and Nano‐mechanical Material Properties Impacting Ex Vivo Expansion and Differentiation of Hematopoietic and Mesenchymal Stem Cells

Jiang

Papoutsakis

2012

Adv Healthcare Materials

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The ability of stem cells to self-renew with minimal or no differentiation and, when appropriately cued, to give rise to many types of progenitor and mature cells, is the basis for applications in regenerative and transfusion medicine, but also in drug discovery and in vitro toxicology. Inspired by the complex interactions between stem cells and their microenvironment, the so-called stem-cell niche, the properties of supporting biomaterials, including surface biochemistry, topography (type, size, organization, and geometry of nanostructures), and mechanical properties, have been identified as important determinants of stem-cell fate in vitro. 3D culture environments that could recapitulate the complexity of the in vivo stem-cell microenvironment could further expand the complexity and repertoire of engineered environments with exciting translational applications. Herein, the material aspects that affect the expansion and differentiation fate of adult hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), two powerful cell types that co-reside in the bone-marrow niche, but with distinct, sometime complementary, differentiation fates, properties, and translational applications, are examined. Although MSCs are adherent cells and, in contrast, HSPCs are non- or weakly adherent cells, both can sense and respond to material properties, including surface (bio)chemistry, ECM composition, topography, and matrix elasticity, possibly through similar molecular mechanisms.

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Micromarrows—Three-Dimensional Coculture of Hematopoietic Stem Cells and Mesenchymal Stromal Cells

Cited by 59 publications

References 27 publications

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

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

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

Stem‐Cell Niche Based Comparative Analysis of Chemical and Nano‐mechanical Material Properties Impacting Ex Vivo Expansion and Differentiation of Hematopoietic and Mesenchymal Stem Cells

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