Bone Marrow Microenvironment-On-Chip for Culture of Functional Hematopoietic Stem Cells

Hematopoiesis takes place in the bone marrow and is supported by a complex cellular and molecular network in the bone marrow microenvironment. Commonly used models of the human bone marrow microenvironment include murine models and two-dimensional and three-dimensional tissue cultures. While these model systems have led to critical advances in the field, they fail to recapitulate many aspects of the human bone marrow. This has limited our understanding of human bone marrow pathophysiology and has led to deficiencies in therapy for many bone marrow pathologies such as bone marrow failure syndromes and leukemias. Therefore, we have developed a modular murine bone marrow microenvironment-on-chip using a commercially available microfluidic platform. This model includes a vascular channel separated from the bone marrow channel by a semi-porous membrane and incorporates critical components of the bone marrow microenvironment, including osteoblasts, endothelial cells, mesenchymal stem cells, and hematopoietic stem and progenitor cells. This system is capable of maintaining functional hematopoietic stem cells in vitro for at least 14 days at frequencies similar to what is found in the primary bone marrow. The modular nature of this system and its accessibility will allow for acceleration of our understanding of the bone marrow.


INTRODUCTION
The adult hematopoietic system arises from the hematopoietic stem cell (HSC) that resides in and is supported by the HSC niche within the bone marrow. The HSC niche is a complex, interconnected network of cellular and molecular components within the bone marrow microenvironment (BMME) (Frisch, 2019). A major challenge that hampers progress in the field of the BMME is the lack of a reliable model system that can recapitulate the human bone marrow. Murine models have been used extensively because they allow for in vivo studies and powerful genetic tools. However, mouse models often fail to recapitulate the human environment. Conventional 2D cultures are another common approach used to investigate the human BMME for the study of normal hematopoiesis and hematopoietic malignancies (Congrains et al., 2021). However, 2D cultures lack the ability to completely elucidate bone marrow pathophysiology and are typically limited to 2-3 different cell types. Overall, the inadequacies of conventional preclinical models contribute to a relatively poor understanding of the human BMME and the large attrition rates of Phase II and III of clinical trials (Arrowsmith and Miller, 2013).
In recent years, in vitro 3D models of various organ systems have been developed to facilitate basic biology research and drug development and screening. The development of these preclinical disease models is beneficial in understanding complex microenvironmental interactions with the potential implications of discovering novel treatment strategies (Duarte et al., 2018). The three-dimensional culture strategy is advantageous as it can provide an avenue to recapitulate the complex tissue microstructures while reducing the exhaustive requirements of animal models (Huh et al., 2011;Low and Tagle, 2017a;Low and Tagle, 2017b;Osaki et al., 2018;Kelm et al., 2019;Low et al., 2020). Therefore, a 3D microphysiological system will advance the field of BMME research by providing a viable platform for applications in mechanistic studies and drug discovery.
Several groups have shown the use of fibrin-based hydrogels in microfluidics platforms and the incorporation of a dynamic vasculature component were able to sustain HSCs in vitro (Chou et al., 2020;Nelson et al., 2021). Synthetically derived scaffolds such as PEG crosslinked with MMP-degradable peptide or tethered with adhesive ligands show promising evidence of HSC sensitivity towards culture dimension and flow conditions (Bray et al., 2017;Rödling et al., 2017). Although these developments provide valuable insights into establishing niche incorporated models, the outcomes of these models are limited to studying myelotoxicity towards drugs and radiation rather than insights into the mechanisms of HSC/niche interactions. Most of the current models lack major BMME components such as osteoblastic cells. Osteoblasts are important in establishing the endosteal HSC niche compartment, regulating bone remodeling and promoting the survival of lymphoid progenitors (Duarte et al., 2018). Our group and others have shown that leukemic cell interactions with the BMME, specifically through osteoblastic crosstalk, can drive leukemic progression and development (Frisch et al., 2012;Staversky et al., 2018;Ackun-Farmmer et al., 2021). Therefore, it is crucial to incorporate the osteoblastic components in addition to the vascular niche to generate a reliable 3D model of the BMME.
In this study, we generated a 3D model of the murine BMME with functional osteoblastic, endothelial, and stromal cell compartments to recapitulate the endosteal niche that is favorable for HSC survival. We used a commercially available microfluidics system, the Human Emulation System from Emulate Bio, and fibrin hydrogel scaffolding to culture HSC and performed competitive transplant assays to evaluate the self-renewal property of BMME-on-chip grown HSCs. The development of this preclinical model is valuable for the study of BMME pathologies, BMME-hematopoietic cells crosstalk, and identifying novel therapeutic targets.

MURINE BMME-ON-CHIP
Our goal was to generate a BMME-on-chip with niche components that are reliable, cost-effective, and easily adopted across research laboratories ( Figure 1). The development of inhouse microfluidics models is beneficial in generating complex architecture for in vitro systems; however, it can be timeconsuming and limited to a specific laboratory. In this study, we utilized Emulate Chip S-1 ™ to provide the geometry and mechanical support to culture cells. The chip contains two channels separated by a semi-porous membrane which allows for the physical separation of cellular compartments. The chip is assembled with a housing system, Pod ™ , that contains input and output reservoirs for both channels and is connected to a module that automatically regulates fluid flow in each channel. The validated setup allows for efficient culturing of cells without the need for frequent media changes, troubleshooting flow modules, or optimizing microfluidics architecture.
We have cultured bone marrow stromal cells (BMSC) isolated from wild type C57BL/6 J mice into the apical channel of Chip S-1 to create a supportive osteoblastic layer ( Figure 2). Upon 14 days of culture under a continuous flow rate of 30 μL/h with osteoblast differentiation media, robust differentiation and mineralization were observed in chips, as shown by positive alkaline phosphatase and Von Kossa staining and quantification ( Figure 2B). On D14, we introduced mouse endothelial C166 cells on the basal surface of the membrane to generate a supportive vascular endothelial monolayer ( Figure 1C). C166 cells completely occupied the membrane of the channel at D2 and surrounded the walls of the channel by D7, as indicated by positive nuclei staining (DAPI, Figure 2D). On D7, we found robust endothelium formation as shown by cobblestone morphology and tight junction protein expression, zonula occludens-1 (ZO-1) ( Figure 2E). On D14, a cell-laden fibrin hydrogel was introduced in the apical compartment containing BMSC and whole bone marrow cells. The chip was then subjected to a culture regime of supplemented endothelial media in the basal channel at 30 ul/hr. The chip was grown for 7 days before harvesting on D21 for analysis ( Figure 1B).
We expect that the BMME-on-chip can provide a conducive environment for marrow cell culture due to the multifaceted culture system. In particular, we are interested in investigating the fate of hematopoietic stem cell and progenitor cells (HSPC) within the marrow compartment after 7 days of chip culture. Therefore, on D21, we harvested cells from BMME-on-chip and control mice BM and stained them for lineage (Gr1, CD3e, B220, and Ter119) and HSPC (Sca1 and cKit) markers 7 ( Figure 3A). HSPC (LSK, Lin−Sca+cKit+) population was preserved in healthy BMME-on-chip after 7 days of culture at~0.3% of total cells. This is two-fold greater than levels to in vivo of~0.15% ( Figure 3B). The data strongly suggest that BMME-on-chip is a reliable surrogate for HSPC maintenance in vitro.
HSPC are self-renewing and multipotent cells able to reconstitute the BM and differentiate into mature blood cells. Therefore, the standard functional analysis of HSPC is via a mouse BM transplantation model, whereby experimental HSPC are transplanted into myeloablated mice with the addition of competitor cells as an internal control ( Figure 4). Sorted LSK cells from C57BL/6 J mice (CD45.2+) were grown in chips with CD45.2+ BMSCs for 14 days ( Figure 4A). The whole content of each chip was mixed with whole BM cells from CD45.1+ PepBoy B6 mouse as a competitor and transplanted into a lethally irradiated CD45.1+ PepBoy B6 mice . Equal numbers of CD45.2+ freshly isolated bone marrow cells were also mixed with CD45.1+ BM cells and transplanted into lethally irradiated CD45.1+ recipients as controls. We analyzed peripheral blood (PB) cells via flow cytometry at multiple timepoints after the transplant. We found that CD45.2+ chip LSK were able to successfully engraft in recipient mice at similar levels compared to control HSPC from whole bone marrow ( Figure 5). In particular, chip LSK gave rise to~40-60% of total PB cells, which include myeloid populations, B lymphocytes, and T lymphocytes ( Figure 5B). To confirm multilineage differentiation capacity, we compared the frequency of myeloid and lymphoid cells that were derived from CD45.2+ cells in primary recipients. CD45.2+ cells harvested from the BMME-on-chip generated myeloid cells at similar frequencies compared to cells harvested from freshly isolated bone marrow (C). At 28-weeks post-transplant, myeloid frequencies were at 20% in peripheral blood of both chip and mouse recipients (D), while lymphoid populations were at~80% (D-E). Thus, our findings suggest that chip-derived cells have the equivalent potential for generating myeloid and lymphoid cells to the mouse control group.
Bone marrow engraftment was analyzed by detecting chimerism of CD45+ HSPC populations at a very late timepoint, 28-weeks post-transplant. Flow cytometry was used to quantify chimerism in LSK population and lineagecommitted myeloid progenitors (Lin−cKit+) and lymphoid precursors (Lin−Sca1+) ( Figures 6A-E). Chimerism was observed in all three populations in the majority of the mice transplanted with HSPC from bone marrow and chip ( Figures 6H-J). In particular, CD45.2+ donor cells were observed at a high percentage in LSK populations of control recipients M1, M4, and M5 and in chip recipients C1, C4, and C5 ( Figure 6H). All of the five control recipients showed engraftment of CD45.2+ donor cells, with M2 and M3 having a majority of the repopulated LSK derived from donor cells. On the other hand, two out of the five chip recipients showed low to no repopulation of LSK by donor cells. Myeloid and lymphoid committed cells also showed chimerism as indicated by CD45.2+ cells in control and chip recipients ( Figures 6I and J).
HSPC subpopulations were further analyzed within the LSK cells in recipient mice with high chimerism (M1, M4, M5, C1, C4, and C5) (Figure 7). Our findings showed CD45.2+ repopulation of myeloid-biased (MPP2 and MPP3) and lymphoid-biased (MPP4) multipotent progenitor populations in chip recipients at similar levels to control recipients (Figures 7B-D and G-I). In addition, CD45.2+ short-term and long-term repopulating HSC (ST-HSC) and (LT-HSC) were also observed at high levels, 40% for both groups, in chip recipients. Overall, the majority of mice transplanted with HSPCs obtained from BMME-on-chip cultures maintained similar distribution of HSPC subpopulations even through this very long-term engraftment.
To investigate long-term HSPC function, a secondary transplant assay was performed by injecting bone marrow cells isolated from primary recipient mice into myeloablated secondary recipients ( Figure 8A). PB was analyzed at 4-and 8-weeks post-transplant. Consistent with engraftment data in the bone marrow, only C1, C4, and C5 samples from the chip group showed engraftment of CD45.2+ cells at 4-and 8-weeks ( Figures  8B and C). Overall, CD45.2+ chip-derived cells comprised approximately 20% of total live cells at 4-and 8-weeks posttransplant ( Figure 8D). Although the mouse group showed higher trends of engraftment, the comparison showed no statistical significance between the groups. Importantly, longterm differentiation of HSPC into mature blood populations was maintained as indicated by monocyte and lymphocyte populations that were CD45.2+ post-transplant (Figures 8E and F).
Our findings currently showed that chip grown HSPC are selfrenewing and capable of regenerating the hematopoietic system, which current 3D systems have not been able to demonstrate. This reliability of the chip to provide a permissive microenvironment for HSC maintenance makes it a valuable platform for future work in studying HSPC interactions with the BMME and BM pathologies. Here, we present step by step methodologies to generate BMME-on-chip, including protocols for Emulate Chip preparation, cell culture, MSC isolation, chip characterization, and competitive transplant assay.
3) Place serum in a 56°C water bath for 30 min and swirl the bottle every 5 min. 4) Make aliquots of heat-inactivated serum and store at −20°C until use.
3) Vacuum filter complete media using a 500 mL vacuum filtration flask. 4) Make 50 mL aliquots of media in conical tubes. 5) Warm up to 37°C prior to use or store in a fridge for short term use or in a freezer for long term use.
Osteoblast Differentiation Media 1) Make 1M stock solution of glycerol 2-phosphate disodium salt hydrate in sterile water. Store stock in smaller aliquots in a −20°C freezer. 2) For 200 mL of differentiation media, combine 10 mg of L-ascorbic acid, 2 mL of 1M thawed glycerol solution, and 200 mL of complete media. 3) Filter media using a 250 mL vacuum filtration system.
Warm media to 37°C before use or store in a freezer or fridge. The media will have a final concentration of 50 μg/ mL of L-ascorbic acid and 10 mM of glycerol 2-phosphate disodium salt hydrate. 3) Gas equilibrate with 0.45 μm Steriflip-HV and warm up media for at least 1 h at 37°C prior to use with chips.
Endothelial Media (10% FBS + 1% PS) for C166 Culture 1) For 500 mL of complete endothelial media, combine thawed 5 mL Pen-Strep, 50 mL of heat-inactivated FBS, and 445 mL of DMEM. 2) Warm up to 37°C prior to use or store at 4°C for short term use.
Supplemented DMEM Media 1) For 50 mL of media combine complete endothelial media (10% FBS +1% PS) with the following concentration of cytokines: EPO 20 ng/mL, G-CSF 1 ng/mL, FLT3L 100 ng/ mL, TPO 100 ng/mL, SCF 50 ng/mL, IL-3 10 ng/mL, and IL-6 10 ng/mL. 2) Warm up to 37°C prior to use or store in a fridge for short term use or in a freezer for long term use. 3) Gas equilibrate with 0.45 μm Steriflip-HV and warm up media for at least 1 h at 37°C prior to use with chips.
Fibrin Gel Components 1) Fibrinogen: resuspend at 25 mg/mL in sterile water and make aliquots of 100 μL or 200 μL. Store in a −20°C freezer. 2) Aprotinin: resuspend at 1 mg/mL in sterile water and make aliquots of 12.5 μL or 25 μL. Store in a −20°C freezer. 3) Thrombin: resuspend at 1 U/μL in sterile water and make aliquots of 1 μL. Store in a −20°C freezer. 4) Collagen Type I solution: make aliquots of 10-25 μL. Store in a fridge at 4°C.

Chip Digestion Solution
1) Warm up complete DMEM in a 37°C water bath for at least an hour. 2) Prepare 5 mL of digestion solution with final concentrations of 1 mg/mL nattokinase, 25 mM HEPES, and 1 mg/mL collagenase type 1.  3) Centrifuge and resuspend the cells in complete media to obtain a cell concentration of 2 × 10 6 cells/ml. 4) Seed 3 ml of cell solution in each well of a tissue culture treated 6-well plates to obtain a seeding concentration of 6 × 10 6 cells/ well or 6.25 × 10 5 cells/cm 2 . 5) Grow cells for four days untouched in a cell incubator at 37°C, 5% CO 2 , and 95% humidity to let the BMSCs adhere. 6) On day 4, replace media with warm complete media to remove non-adhered cells, dead cells, and debris. 7) Expand BMSCs for another 7-10 days before using use. Replace media every 2-3 days.  2) Once activated, each cell compartment is added in a sequential manner according to the workflow timeline in Figure 1B.   . 14) Set the flow settings to 30 μL/h in the apical channel and no flow in the basal channel. 15) Differentiate cells for 14 days while checking for air bubbles in the channels every 2-3 days. Clear any air bubbles with gas equilibrated media. 16) Add gas equilibrated media into the apical channel inlet reservoirs every 2-3 days and remove the content of the outlet reservoir. Replace 300 μL of media into the reservoir to avoid drying out.

Day 0: (II) BMSC Culture Preparation
Isolate and culture bone marrow cells from C57BL/6 J to obtain BMSC on Day 14 for cell encapsulation. Follow protocols for primary bone marrow cell isolation and BMSC culture.
Day 10: C166 Cell Line Culture 1) Warm up complete endothelial media to 37°C in a water bath.
Day 14: (I) Endothelial Cell Seeding 1) Trypsinize C166 endothelial cells in T-75 flasks using 5 mL of 0.25% Trypsin-EDTA for 3 min in a cell culture incubator. 2) Neutralize the trypsin by adding 20 mL of gas equilibrated complete endothelial media. Gently pipette the solution to release the cells and centrifuge the cells at 1000 rpm or 500 g. Resuspend the cells in 10 mL of media and count the cells. 3) Seed 25 μL of 2 × 10 6 cells/mL C166 cell solution into the basal channel of the chips similarly according to the BMSC seeding protocol. 4) Invert the chips to allow cells to adhere onto the membrane surface of the basal channel ( Figure 1A). Leave the cells to adhere in the incubator for two hours.
Day 14: (II) Hydrogel Seeding and Coculture 1) Isolate bone marrow cells from 1 C57BL/6 J mouse according to the primary bone marrow cell isolation protocol. Count the cells and resuspend them in gas equilibrated supplemented DMEM media.
2) Trypsinize and collect the BMSC grown for 14 days. Count the cells and resuspend them in gas equilibrated supplemented DMEM media. 3) Thaw fibrin hydrogel components. Calculate the volume of each component needed for the number of chips to be seeded. Each chip will obtain 45 μL of fibrin gel solution containing whole bone marrow cells and BMSC in the apical channel. The final gel solution will have 5 mg/mL of fibrinogen, 25 μg/mL aprotinin, 0.2 mg/mL collagen type 1, and 0.5 U/mL thrombin. 4) To prevent premature crosslinking, prepare the gel solution in two separate mixes. Combine fibrinogen and aprotinin in tube A and collagen type 1 and thrombin in tube B. 5) Combine 6 × 10 4 BMSC and 6 × 10 4 bone marrow cells per chip and resuspend in the amount of media required to bring up the total volume to the desired final volume. Mix the cell solution with tube B. 6) For each chip, separately aliquot the desired volume tube A and tube B containing media with cells and immediately seed into the apical channel of Chip S-1. Check for bubbles. Reseed the apical channel in the presence of air bubbles. 7) Leave the chips in the cell incubator for 30 min to 1 h in a humidified cell culture dish for the gel to fully polymerize. 8) Remove the contents of the inlet and outlet reservoirs of the Pod ™ and fill up 3 mL of gas equilibrated supplemented endothelial media in the basal channel inlet reservoirs. Add about 300 μL of media to the remaining reservoirs. 9) After the gel is polymerized, connect Chip S-1 to Pod ™ and Zoë ™ . Setup flow at 30 μL/h in the basal channel where the endothelial cell monolayer is present. 10) Culture chip for another 7 days. Check for bubbles and replace the media every 2-3 days.
2) Disconnect Chip S-1 from the Zoë ™ and plug the outlet ports of the apical and basal channels with a 200 μL pipette tip. 3) Add 100 μL digestion solution to the apical channel and 50 μL digestion solution to the basal channel. Leave pipette tips in the inlet port. 4) Incubate the chips for 1 h in the incubator. 5) After 1 h, collect the contents of the apical and basal channel into a microcentrifuge tube. 6) Trypsinize the channels with 0.25% Trypsin-EDTA for 5 min at 37°C to collect the remaining cells on the membrane surfaces. Check for detachment under a light microscope. 7) Collect trypsin digest into the same collection tubes and add 1 mL of media to neutralize the trypsin. 8) Centrifuge the cell suspension to remove the digest media. 9) Resuspend cells in 1 mL of media and count the cells. 10) The cells are now ready for appropriate analysis steps.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org June 2022 | Volume 10 | Article 855777 Flow Cytometry 1) C57BL/6 J marrow cell control. Isolate cells from each mouse using the previous cell isolation protocol. Cells can be stored in liquid nitrogen. 2) Resuspend the cells in a 5 mL 1 × RBC lysis buffer for 5 min at room temperature. 3) Wash with 5 mL FACS buffer. Centrifuge the cells and decant the supernatant. 4) Resuspend the cells in FACS buffer and set aside 1 × 10 6 of isolated C57BL/6 J cells for DAPI and unstained compensation controls. 5) Prepare the primary lineage staining solution with the appropriate antibodies (Supplementary Table S1).
Prepare the desired volume of master mix for 1 μL of each antibody solution per sample. 6) Centrifuge the harvested cells and controls in FACS tubes.
Remove the supernatant. 7) Add 100 μL of lineage-biotin staining solution to each sample and leave the cells at 4°C for 25 min. In the meantime, prepare fluorophore-conjugated antibodies staining solution Supplementary Table S1. Generate chips with osteoblastic layer and endothelium layer according to BMME-on-chip preparation protocol: Day 14 to Day 14 (I).
Control Group: Each recipient will receive a number of C57BL/6 J bone marrow cells (CD45.2+ donor) equivalent to the average number of cells obtained from the chips and 1 × 10 5 CD45.1+ competitor cells.
Each mouse will receive a cell solution of 100 μL in FACS buffer.
2) Perform secondary irradiation of the ten recipient mice with 5Gy irradiation. Alkaline Phosphatase and Von Kossa Staining 1) To analyze mineralization for osteoblastic differentiation on chips, fix chips by adding 10% neutral formalin buffer into the inlet of the apical (50 μL) and basal channels (20 μL). Fix cells at room temperature for 30 min. 2) Wash the cells with 1x distilled water for 5 min. Repeat 2-3 times. 3) Add prepared alkaline phosphatase staining solution into the apical and basal channel of each chip. Incubate for 45 min at room temperature. 4) Rinse in distilled water 3-4 times and leave in distilled water for 1 h. 5) Stain with 2.5% silver nitrate solution of 30 min. 6) Aspirate the solution and rinse with distilled water 3 times. 7) Image cells under a microscope or store them at 4°C. For the abovementioned experiments, images were taken using Biotek Cytation 5 Cell Imagine Multimode Reader at 10 × magnification with color brightfield. Mosaic imaging and stitching were automated using Biotek Gen5 imaging and analysis software.

Image Analysis Using ImageJ
Image analysis of the alkaline phosphatase and Von Kossa stained chips was performed using ImageJ software, as described in Supplementary Figure S1. Immunofluorescent staining and imaging were performed as described in Supplementary Figure S2. Image analysis of ZO-1 and DAPI staining was performed in ImageJ, as described in Supplementary Figure S3.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 9 statistical analysis software. For all quantitative results, groups are reported as mean ± standard deviation (SD). For comparisons between two groups, statistical analysis was performed using student's unpaired two-tailed t-test for normally distributed. Otherwise, unpaired and nonparametric multiple t-tests were performed with Mann-Whitney post-test. For comparisons between two groups across timepoints ( Figure 5), statistical analysis was performed using restricted maximum likelihood (REML).

ANTICIPATED RESULTS
We anticipate that the modular nature of this BMME-on-chip will allow for rigorous analysis of individual cellular components of the bone marrow microenvironment in a model that more closely recapitulates the in situ HSC niche. The modular nature of this model and the ability to use different cell populations from unique sources and genetic backgrounds is a powerful tool. We anticipate that this system will be used to clarify the roles of individual cell populations of the BMME during hematopoietic homeostasis, as well as during aging, and in pathological states such as leukemia and multiple bone marrow failure syndromes.

LIMITATIONS
The described murine BMME-on-chip provides validation that an in vitro system is capable of maintaining multi-potent and self-renewing HSCs for at least 14 days. Competitive transplantation of HSCs into an immune competent recipient is the gold standard for quantifying HSC function. As this analysis is not possible with the use of human cells, this represents a strength of the murine system in validation prior to the use of a fully human BMME-onchip. However, it also represents a limitation as mouse models do not always recapitulate the human environment. Previously published BMME-on-chip devices that use human tissue have demonstrated the ability to maintain phenotypic HSPCs (Chou et al., 2020). Therefore, it is likely that the functional ability to self-renew and repopulate the hematopoietic system of a myeloablated recipient that we report here in the murine model is translatable to a human environment. While our murine BMME-on-chip includes multiple key components of the bone marrow HSC niche, it is not fully representative of the physiologic bone marrow. For example, osteoclastic bone resorption is an ongoing process in vivo and is a component not included in our system. In addition, osteocytes embedded in bone are the most abundant bone cell in vivo and are not present in our BMME-onchip (Frisch, 2019). The relative importance of these cells and Frontiers in Bioengineering and Biotechnology | www.frontiersin.org processes for the regulation of HSCs are not well-established, and thus the implication of their absence in our system is not known . However, the ability of our BMME-on-chip to maintain a fully functional HSC population suggests that they are not required for HSC maintenance.

CONCLUSION
In leukemia such as acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), leukemic cells can interact with the surrounding stroma to create a favorable environment for leukemia stem cell LSC maintenance and expansion of leukemic cells (Frisch et al., 2012;Shafat et al., 2017;Behrmann et al., 2018;Staversky et al., 2018;Xu et al., 2021). The BMME is made up of cellular and noncellular components such as BMSC, endothelial cells, osteoblasts, osteoclasts, fibroblasts, adipocytes, macrophages, and extracellular matrix that provide crucial signals for the homeostatic regulation of hematopoietic systems. Leukemia has been shown to interact with the BM niche by modulating endothelial activation, establishing crosstalk with fibroblasts, and inhibiting osteoblasts (Ryningen et al., 2005;Frisch et al., 2012;Pezeshkian et al., 2013;Krevvata et al., 2014;Shafat et al., 2017;Staversky et al., 2018). In particular, our group and others have shown that leukemic cell interaction with osteoblasts is an important component in sustaining leukemic burden. However, the interactions of the HSC niche with bone marrow pathologies are poorly understood . AML is the most common form of leukemia that arises in the bone marrow . The standard therapy for AML of combined daunorubicin and cytarabine administration, termed 7 + 3 induction therapy, which was first employed in the 1970s, is still the predominant upfront therapy for most AML patients and gives rise to poor outcomes with an overall 5-years survival rate of <30% (Roussel et al., 2020;Kantarjian et al., 2021). Therefore, a better understanding of the role of the BMME in AML is critical for the improvement in therapy and patient outcomes.
The use of a BMME-on-chip that recapitulates many of the cellular interactions that occur in the BMME in vivo will allow for rigorous analysis of the BMME during hematologic malignancies. A murine environment, like the one in the BMME-on-chip described herein, will validate that phenotypes identified in vivo murine leukemic models can be recreated in an in vitro environment, and suggest that the in vitro environment of a similar human BMME-on-chip will recreate human bone marrow phenotypes that are not possible to directly observe in vivo. Therefore, this BMMEon-chip microphysiological platform will serve as a critical pre-clinical model for the identification of novel therapeutic targets.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the University Committee on Animal Resources, University of Rochester.

AUTHOR CONTRIBUTIONS
AS performed experiments and wrote the manuscript. ML performed experiments. CS performed experiments. BF oversaw all experiments and wrote and edited the manuscript.

FUNDING
The experiments described were funded by the Department of Laboratory Medicine and the Wilmot Cancer Institute at the University of Rochester Medical Center.