Platelet bioreactor-on-a-chip

Abstract

• We have developed a biomimetic microfluidic platelet bioreactor that recapitulates bone marrow and blood vessel microenvironments.• Application of shear stress in this bioreactor triggers physiological proplatelet production, and platelet release.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. (Blood. 2014;124(12):1857-1867) IntroductionAlthough 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 apheresisequivalent 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, donorindependent 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. 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 10 122 PLTs per CD34 1 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 c...

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein

“…Proplatelets contain beaded swellings and platelet-sized tips that are morphologically and structurally identical to in vivo proplatelet production, as documented by Two-Photon microscopy. 5,23,24 Proplatelets formed by MKs within the platelet bioreactor elongated, on average, at a rate of 30 mm/minute ( Figure 5A; supplemental Video 12; supplemental Figure 1E; Thon et al 12 ), which is a much higher rate than the average rate measured for proplatelet formation in culture 11 and more accurately reproduces proplatelet extension rate in vivo. Next, we treated mouse MKs in the bioreactor with drugs that interfere with different aspects of microtubule function.…”

“…12 For static cultures, isolated MKs were pipetted into chambers formed by mounting a glass cover slide coated with 3% BSA onto a 10-mm petri dish with a 1-cm hole, as previously described, 7 and cultured for 24 hours. Both static and shear stress cultures were maintained at 37°C and 5% CO 2 …”

“…12,15 Proplatelet elongation was quantified between the individual frames of time-lapse movies taken every minute. The length of proplatelets was measured in the individual frames, using ImageJ.…”

“…To determine how these physiologically relevant shear stresses affect microtubule sliding, we studied proplatelet movements made when isolated fetal liver cell-derived mouse MKs were infused into a biomimetic platelet bioreactor that recapitulates sinusoidal blood vessel architecture and flow profile. 12 Briefly, this bioreactor comprises 2 parallel microfluidic channels (an upper and a lower channel) that are separated by a series of columns spaced 1 to 2 mm apart. MKs in culture media are infused in the upper channel, and flow is directed through the gaps between the columns, so that the media exits from the lower channel.…”

Transcriptional Regulation of Platelet Formation: Harnessing the Complexity for Efficient Platelet Production In Vitro

“…Maximising platelet production in vitro from these MKs will be a condition without which production of platelets on the scale necessary for a clinically relevant product (each platelet concentrate contains 3 × 10 11 platelets) will remain a dream. The apparent shortfall in platelet formation might be due to our inability to re-create the niche signal (including shear) necessary for efficient proplatelet formation and platelet release, although several approaches using 3D bioreactors have been published to that effect [212][213][214]. It is also worth mentioning that the evidence that platelets released in vitro are functional has been gathered using descriptive rather than quantitative assays with usually no comparison to platelets freshly isolated from donors.…”