Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs.

This article describes the development of a scalable, third-generation, human-induced pluripotent stem cell-derived MK-compatible PLT bioreactor that uses biologically inspired engineering to fully integrate the major chemical and physical components of the BM stroma under high-resolution live-cell microscopy. This work constitutes a major advance in PLT biology by providing a window into the complex physiology of thrombopoiesis, and a biomimetic platform to elaborate the physiological drivers of proPLT production, accelerate PLT release, and generate a donor-independent source of functional human PLTs for infusion.

In vivo, megakaryocytes (MKs) PLT progenitors sit outside blood vessels in the bone marrow (BM) and extend long, branching cellular structures designated proPLTs into the circulation from which PLTs are released. 11-15 Nearly 100% of human adult MKs must produce ∼10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ∼10% of human MKs initiate proPLT production in culture. This results in yields of 10 1 − 2 PLTs per CD34 + cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation cell culture approaches have provided further insight into the physiological drivers of PLT release, they have been unable to recreate the entire BM microenvironment, exhibiting limited individual control of extracellular matrix (ECM) composition, 19 , 20 BM stiffness, 21 endothelial cell contacts, 22 , 23 and vascular shear stresses, 24 , 25 and have been unsuccessful in synchronizing proPLT production, resulting in nonuniform PLT release over a period of 6 to 8 days. 26 Moreover, the inability to reproduce the BM microenvironment ex vivo, and resolve physiological proPLT extension and release by high-resolution live-cell microscopy, has significantly hampered efforts to study the cytoskeletal and signaling mechanics of PLT production. Biomimetic platforms that model human BM are needed to support drug development and establish new treatments for thrombocytopenia.

Although platelets (PLTs) play critical roles in hemostasis, 1 angiogenesis, 2 and innate immunity, 3 PLT production remains poorly understood. Consequently, PLT units are derived entirely from human donors, despite serious clinical concerns owing to their immunogenicity and associated risk of sepsis. 4 More than 2.17 million apheresis-equivalent PLT units are transfused yearly in the United States 5 , 6 at a cost of >$1 billion per year. Although demand for PLT transfusions has increased markedly in the past decade, a near-static pool of donors and a 5-day PLT unit shelf life resulting from bacterial contamination 7 and storage-related PLT deterioration, 8 have resulted in significant PLT shortages. 9 Furthermore, artificial platelet substitutes have failed to replace physiological platelet products. 10 An efficient, donor-independent PLT bioreactor capable of generating clinically significant numbers of functional human PLTs is necessary to obviate risks associated with PLT procurement and storage, and help meet growing transfusion needs.

Rectangular microfluidic channels 30 µm deep for mouse fetal liver culture-derived–MKs and 10 µm deep for human-induced pluripotent stem cell culture (hiPSC)-MKs were fabricated using soft lithography as previously described. 28 AutoDesk software in AutoCAD was used to design the desired two-dimensional pattern and printed on a photolithography chrome mask. The silicon wafer (University Wafers, Boston, MA) was spin coated with SU-8 3025 photoresist (Michrochem, Newton, MA) to a 30 µm film thickness (Laurell Technologies, North Wales, PA), baked at 65°C for 1 minute and 95°C for 5 minutes, and exposed to UV light (∼10 mJ · cm 2 ) through the chrome mask for 30 seconds. The unbound SU-8 photoresist was removed by submerging the substrate into propylene glycol monomethyl ether acetate for 7 minutes. PDMS was poured onto the patterned side of the silicon wafer, degassed, and cross-linked at 65°C for ∼12 hours. After curing, the PDMS layer was peeled off the mold and the inlet/outlet holes were punched with a 0.75 mm diameter biopsy punch. The microchannels were sealed by bonding the PDMS slab to a glass coverslide (#1.5, 0.17 × 22 × 50 mm; Dow Corning, Seneffe, Belgium) after treatment with oxygen plasma (PlasmaPrep 2; GaLa Instrumente GmbH, Bad Schwalbach, Germany). Samples were infused into the microfluidic bioreactor via polyethylene/2 tubing (Scientific Commodities, Lake Havasu City, AZ) using 1 mL syringes equipped with 27-gauge needles (Beckton Dickinson, Franklin Lakes, NJ). Flow rates of liquids were controlled by syringe pumps (PHD 2000; Harvard Apparatus, Holliston, MA).

PLT bioreactor design. (A) PLT bioreactors are based on custom-built polydimethylsiloxane (silicon-based organic polymer) bonded to glass slides, and are comprised of an upper and lower microfluidic channel separated by a series of columns. (B) The 2 µm gaps separate columns to trap MKs entering the upper channel from crossing into the lower channel upon fluid withdrawal from the outlet of the lower channel. (C) Scanning electron micrograph of bioreactor central channels. (D) Scaled bioreactor design showing typical device operation. Scale bars represent 50 µm (B) and 5 µm (C).

PLT bioreactor design. (A) PLT bioreactors are based on custom-built polydimethylsiloxane (silicon-based organic polymer) bonded to glass slides, and are comprised of an upper and lower microfluidic channel separated by a series of columns. (B) The 2 µm gaps separate columns to trap MKs entering the upper channel from crossing into the lower channel upon fluid withdrawal from the outlet of the lower channel. (C) Scanning electron micrograph of bioreactor central channels. (D) Scaled bioreactor design showing typical device operation. Scale bars represent 50 µm (B) and 5 µm (C).

To ensure efficient gas exchange and support high-resolution live-cell microscopy during cell culture, microfluidic bioreactors were constructed from transparent polydimethylsiloxane (PDM), which is a cell-inert silicon-based organic polymer 27 bonded to glass slides. The microfluidic bioreactor consists of 2 inlet channels containing passive filters used to trap air bubbles and dust, followed by fluid resistors used to dampen fluctuations in flow rate arising during chip operation. The inlet channels meet in a central channel 1300 µm long and 130 µm wide, separated by a series of columns (10 µm wide and 90 µm long) spaced 2 µm apart. For the purposes of clarity the “upper channel” should be construed to represent the top-most channel in the original bioreactor ( Figure 1A-C ) and the central channel in the scaled bioreactor ( Figure 1D ). Likewise, the “lower channel” should be construed to represent the bottom-most channel in the original bioreactor ( Figure 1A-C ) and the side channels in the scaled bioreactor ( Figure 1D ). Primary MKs become trapped in these gaps while moving from the upper channel into the lower channel. Two outlet channels collect the effluent.

Bioreactors were coated with a 0.22 µm filtered (Millipore, Billerica, MA) 10% bovine serum albumin solution for 30 minutes to prevent direct cell contact with glass. Primary MKs and media were infused in the top-left and bottom-left inlets, respectively, at a rate of 12.5 µL/hour using a two-syringe microfluidic pump (Harvard Apparatus, Holliston, MA). When the top-right outlet is closed, both input solutions are redirected out the bottom-right outlet causing primary MKs to trap in the gaps separating the 2 middle channels.

PLT bioreactors were designed to recapitulate the dimensions of human venules29 in the BM (Figure 1A) and are comprised of upper and lower microfluidic channels separated by a series of columns (spaced 2 µm apart) (Figure 1B-C) to model proPLT extension through gaps in vascular endothelium under controlled flow conditions. To scale PLT production the bioreactor was lengthened, and upper and lower microfluidic channels were mirrored as outlined in Figure 1D. Precise bioreactor dimensions are outlined in the supplemental Material (available on the Blood Web site).

Our bioreactor was designed to support integration of micro-channel size, ECM composition, BM stiffness, endothelial cell contact, and hemodynamic vascular shear stress within a single platform device. The upper and lower channels can be selectively coated with ECM proteins to reproduce the biochemical composition of the BM and blood vessel microenvironments (Figure 2A). By blocking the upper right channel output to direct flow across the bioreactor, primary mouse MKs infused along the top channel become sequentially trapped between the columns and extend proPLTs into the lower channel (Figure 2B), recapitulating physiological proPLT extension (white arrow).11,30-32 To model three-dimensional ECM organization and physiological BM stiffness (250 Pa)33 (supplemental Figure 1A-D), primary mouse MKs were infused in a 1% alginate or 3 mg/mL growth factor reduced Matrigel solution. Alginates are naturally derived polysaccharides that are cell-inert and can be modified with RGD-containing cell adhesion ligands to specifically reproduce MK-matrix interactions in the BM, such as glycoprotein (GP)IIb/IIIa receptor binding to fibrinogen.34 Matrigel is a solubilized basement membrane preparation that is comprised primarily of laminin and collagen type IV, which are both known agonists of proPLT production, and is preferred over collagen type I-based hydrogels, which is a known inhibitor of proPLT production.19,20 Hydrogels were polymerized within the microfluidic bioreactor, selectively embedding the MKs in a three-dimensional gel within the upper channel, while retaining vascular flow in the lower channel (0.02 µm fluorescent bead streaking, fluorescein isothiocyanate-dextran fluorescence) (supplemental Figure 1C). MK distance from the lower channel could be tightly controlled by regulating infusion rate and starting/stopping flow on the microfluidic pump (supplemental Figure 1A-B). Alginate did not inhibit proPLT production (supplemental Figure 1E). To further reproduce BM blood vessel physiology, human umbilical vein endothelial cells (HUVECs) were selectively seeded on fibronectin in the lower channel and grown to confluency (Figure 2C). HUVECs were positive for CD31 (biomarker) and DAF-2 DA (nitric oxide, data not shown). MK trapping, BM stiffness, ECM composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts can be combined to reproduce the BM vascular niche in vitro, as is represented with primary mouse MKs in Figure 2D.

Figure 2 View largeDownload PPT PLT bioreactor models major components of BM. Primary mouse MKs are shown. (A) The upper and lower channels can be selectively coated with ECM proteins to reproduce osteoblastic and vascular niche composition. (B) MKs trap at gaps and extend proPLTs into the lower channel (white arrow). MKs can be selectively embedded in alginate or Matrigel gels, modeling three-dimensional ECM organization and physiological BM stiffness (250 Pa). Vascular flow is retained in the lower channel as demonstrated by 0.02 µm fluorescent bead streaking and fluorescein isothiocyanate-dextran fluorescence. (C) HUVECs can be selectively cultured in ECM-coated channels to reproduce blood vessel physiology. (D) MK trapping, BM stiffness, ECM composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts can be combined to reproduce human BM in vitro. (E) Shear rate distribution along the length of the channel. Arrows indicate the magnitude and direction of the velocity field. (F) Fluid shear rates are well-characterized and can be tightly regulated across the bioreactor as a function of flow rate. (G) Regardless of the number of occupied slits, trapped MKs experience physiological shear stresses at gap junctions. Media viscosity is 1.20 mPa · s. Scale bars represent 50 µm (A-D). Figure 2 View largeDownload PPT PLT bioreactor models major components of BM. Primary mouse MKs are shown. (A) The upper and lower channels can be selectively coated with ECM proteins to reproduce osteoblastic and vascular niche composition. (B) MKs trap at gaps and extend proPLTs into the lower channel (white arrow). MKs can be selectively embedded in alginate or Matrigel gels, modeling three-dimensional ECM organization and physiological BM stiffness (250 Pa). Vascular flow is retained in the lower channel as demonstrated by 0.02 µm fluorescent bead streaking and fluorescein isothiocyanate-dextran fluorescence. (C) HUVECs can be selectively cultured in ECM-coated channels to reproduce blood vessel physiology. (D) MK trapping, BM stiffness, ECM composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts can be combined to reproduce human BM in vitro. (E) Shear rate distribution along the length of the channel. Arrows indicate the magnitude and direction of the velocity field. (F) Fluid shear rates are well-characterized and can be tightly regulated across the bioreactor as a function of flow rate. (G) Regardless of the number of occupied slits, trapped MKs experience physiological shear stresses at gap junctions. Media viscosity is 1.20 mPa · s. Scale bars represent 50 µm (A-D).