Supplementary Information. Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro

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1 Supplementary Information Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro Yu-suke Torisawa 1, Catherine S. Spina 1,2, Tadanori Mammoto 3, Akiko Mammoto 3, James C. Weaver 1, Tracy Tat 3, James J. Collins 1,2,4,5, and Donald E. Ingber 1,3,6 1 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 2115; 2 Boston University School of Medicine, Boston, MA 2118; 3 Vascular Biology Program, Departments of Pathology and Surgery Children s Hospital Boston and Harvard Medical School; 4 Howard Hughes Medical Institute, 5 Department of Biomedical Engineering, Boston University, Boston, MA 2115, USA; 6 School of Engineering and Applied Science, Harvard University, Cambridge, MA 2138, USA; Both authors contributed equally.

2 Supplementary Fig. 1. Implantation of PDMS devices filled with bone-inducing materials. PDMS device (two openings) Improved PDMS device (one opening) Skin Muscle Bone-inducing materials (collagen I, DBP, BMP2 and BMP4) were embedded in the central cylindrical cavity of the PDMS device and implanted subcutaneously on the back of a mouse. The PDMS devices were sutured onto the underlying muscle. The device with two openings (left) permitted cellular ingrowth from both the muscle and the adipose layer of the overlying skin. The device was improved (right) by covering the top opening with a solid PDMS layer so that the boneinducing materials only contacted the underlying muscle.

3 Supplementary Fig. 2. Images of engineered bone marrow. 4 wk 8 wk Photographs showing vascularized bone containing marrow in 4 and 8 weeks following implantation (bar, 2 mm).

4 Normalized (wt. %) Supplementary Fig. 3. The engineered bone exhibits normal trabecular microarchitecture and mineralization. A B BSE BSE Ca Ca 4wk P P 8wk Vertebra 8wk Vertebra C 8 7 Calcium Phosphorus 5 3 Calcium P 1 Vertebra 8wk (A) Three-dimensional reconstruction of micro-computed tomography (micro-ct) data from 4 (top) and 8 weeks (middle) following implantation. Note: the ultrastructural similarities in both cortical and trabecular bone organization between the 8 weeks after implantation and a normal mouse vertebra (bottom). Analysis suggests that the trabecular bone underwent normal remodeling between 4 to 8 weeks to produce a finer bone network with increased marrow space. (B) Compositional backscattered scanning electron (BSE) micrographs and elemental mapping using energy dispersive x-ray spectroscopy (EDS) of cross sections of a mouse vertebra (left) and an 8 weeks following implantation (right). Calcium and phosphorous EDS maps reveal similar uniform mineralization in both samples (bar, 1 mm). (C) Quantitation of the EDS analysis results analyzing the relative calcium and phosphorus concentrations in the samples from (B) confirm indistinguishable elemental compositions in the compared to living bone. Error bars represent S.E.M.

5 Supplementary Fig. 4. Immunofluorescent analysis of bone marrow niche components. / CD31 B / Nestin C / LepR D Femur / CD31 E Femur / Nestin F Femur / LepR DAPI DAPI A Confocal fluorescence micrographs showing cross sections of (A-C) stained with CD31, Nestin and Leptin receptor (LepR), compared with cross-sections of bone marrow within the mouse femur (D-F). Blue fluorescent images show DAPI staining of nuclei. Note: Nestin and LepR appear in perivascular regions while CD31 is limited to the vascular endothelium in both native marrow and the. Bars, 25 m.

6 Distribution (%) of Lin - Sca1 + ckit + cells Supplementary Fig. 5. Distribution of CD34 cells within the HSC (Lin - Sca1 + ckit + ) compartment. 8 CD34- CD CD34- CD wk 8wk mbm Distribution of CD34 - (black) and CD34 + (gray) cells within the Lin - Sca1 + ckit + (HSCs) population in 4 wk, 8 wk and freshly harvested mbm (n = 5). Error bars represent S.E.M.

7 Distribution (%) Supplementary Fig. 6. Abundance of LT-HSCs in LSK (Lin - Sca1 + ckit + ) compartment. CD15+CD48-LSK CD15-CD48+LSK wk mbm CD15+CD48- CD15-CD48+ Abundance of CD15 + CD48 - LSK (black) and CD15 - CD48 + LSK (gray) cells in 8 wk and freshly harvested mbm (n = 5). Error bars represent S.E.M.

8 Viability (%) Supplementary Fig. 7. In vitro culture of Series L/min Viability of hematopoietic cells isolated from after 4 days of culture on-chip under different flow rates (n = 5). Error bars represent S.E.M.

9 Supplementary Fig. 8. Histology and immunohistochemistry of after 4 days of culture on-chip. A B (A) Histological H&E-stained section of cultured on-chip for 4 days (bar, 1 mm). (B) Immunohistochemical analysis of CXCL12 in after 4 days of culture (bar, 25 m). Image demonstrates the presence and normal localization of CXCL12- expressing stromal cells lining the engineered bone marrow.

10 Lin-CD34 Lin-CD135 Side scatter Lin-Sca1 Lin-CD48 Lin-CD34 Lin-CD135 Side scatter Lin-Sca1 Lin-CD48 Supplementary Fig. 9. Characterization of bone marrow cells after 4 days in culture. mbm Day 4 (Stroma-supported culture) Lin Lin-cKit Lin-CD Side scatter Side scatter Day 4 (Microfluidic culture) Lin Lin-cKit Lin-CD Side scatter Side scatter Flow cytometry data shows Lineage-depleted cells (Lin-), HSCs (Lin - Sca1 + ckit + ), longterm HSCs (Lin - CD15 + CD48 - ), and progenitor populations (Lin - Sca1 + ckit -, Lin - Sca1 - ckit +, Lin - CD34 +, Lin - CD135 + ). Mouse bone marrow (mbm) cells were freshly isolated from adult mouse femur and cultured on a stromal cell layer in vitro for 4 days (mbm, Day 4). The was cultured in the microfluidic device for 4 days (, Day 4). Numbers inside individual gates indicates percentage of total cells. About 4 million cells were harvested from the bone marrow of each at the time of harvest.

11 Viability (%) Supplementary Fig. 1. In vitro culture of mbm mbm D4 mbm D7 D4 D7 Viability of cells isolated from uncultured mouse bone marrow (mbm, black) from adult femur, femur bone marrow cultured for 4 and 7 days on a stromal-supported culture system (mbm D4, mbm D7), cells analyzed immediately after surgical removal (, gray), and cells after 4 and 7 days of culture on-chip ( D4, D7) (n = 7). Error bars represent S.E.M.

12 Normalized to control (%) (CD45 + CD3 +/- CD19 +/- ) Normalized to control (%) (CD45 + Mac1 +/- G1r +/- ) Normalized to control (%) (Lin - Sca1 + ckit + ) Normalized to control (%) (Lin - CD34 + ) Normalized to control (%) (Lin - ckit + ) Supplementary Fig. 11. Radiation toxicity of the bone marrow-on-a-chip. A HSCs B Hematopoietic Progenitors in vivo Dish in vivo Dexter in vivo Dexter in vivo Dexter Radiation (Gy) Radiation (Gy) Radiation (Gy) C Lymphoid D Myeloid in vivo Dexter in vivo Dexter Radiation (Gy) Radiation (Gy) Effects of -radiation (1 or 4 Gy) normalized to control ( Gy). (A) HSCs (Lin - Sca1 + ckit + ), (B) hematopoietic progenitors (Lin - CD34 +, Lin-cKit + ), (C) lymphoid cells (CD45 + CD3 +, CD45 + CD19 + ) and (D) myeloid cells (CD45 + Mac1 +/- Gr1 +/- ) within bone marrow of freshly isolated from femurs of living mice (in vivo), cultured on-chip for 4 days (), or 4 day stroma-supported culture (Dish) ( P <.5; n = 5).