Patent Application
20200123487
The present disclosure relates to the production of platelets from megakaryocytes (Mks). In particular, the present disclosure provides systems and methods for the in vitro production of platelets from Mks using a microfluidic bioreactor having a center flow channel and uniform high-shear micrometer slits. Use of this microfluidic bioreactor enables the continuous production of millions of platelets and facilitates real-time and long-term visualization of proplatelet and platelet generation.
More than 2 million platelet units are transfused each year in the U.S. to treat patients with thrombocytopenia (low platelet counts) or defective platelets. Platelets are small (2-3 μm) anucleate discoids responsible for thrombosis and hemostasis. Platelet units are collected from volunteer donors via apheresis or from the huffy coats of 4-8 whole blood donations. Hospitals depend on a steady supply of platelet donors. Disruptions of this supply together with a 5-day platelet shelf-life can result in critical shortages. In addition, because platelets require room temperature storage to maintain activity, there is risk of bacterial contamination prior to transfusion. The use of a current Good Manufacturing Practices (cGMP) process for platelet production from megakaryocytes (Mks) would allow for better control and characterization of transfusion units, and could transition the supply from fluctuating donors to a steady in vitro process. Finally, culture-derived platelet production could reduce the risk of immunogenic reactions by avoiding the need to provide platelets from multiple donors.
Platelet formation starts from Mks, which undergo extensive cytoskeletal rearrangements to create proplatelets (proPLTs), the precursors to platelets. In the bone marrow, intravital microscopy studies in mice show that Mks directionally extend proPLTs into the blood sinuses, where shear forces (1.3-4.1 dynes/cm2) elongate and fragment proPLTs into preplatelets (prePLTs). High shear stress in the lung capillary bed shears proPLTS and prePLTs into individual platelets, and can also process Mks directly into platelets. The importance of shear forces has led to the experimental use of bioreactors to study and enhance platelet-like-particle (PLP) release from mature Mks. Estimates of platelet production in vivo are typically greater than 1000 PLPs per Mk; in comparison, in vitro studies typically report less than 100 PLPs per Mk, and in many cases, less than 10 PLPs per Mk.
A major challenge in the development of platelet bioreactors is that much remains unknown about the ex vivo initiation and regulation of proPLT formation, as well as how to maximize PUP release. Parallel-plate flow reactors (PPFRs) are the simplest bioreactors that have been used to study proPLT/PLP formation from adhered Mks under high (1800 s−1)13 and low (400 s−1) shear rates. However, it is difficult to carry out long-term analysis of individual Mks due to transient adhesions as Mks roll over the PPFR surface. Improvements to open-channel PPFRs include introducing an array of vWF (Von Willebrand factor)-coated columns in bioreactors. The anchoring of Mks to columns, at a shear rate of 5000 s−1, allowed longer Mk retention for analysis and study of the proPLT formation step. Complex niche bioreactors occupy the other end of the bioreactor spectrum. For example, a 3D silk-based porous microtube surrounded by a silk sponge reproduced the structure of a blood sinus and the bone marrow niche. Using a shear rate of 60 s−1, the system reproduced PLP production in a physiologically relevant environment, but real-time visualization was challenging. Therefore, insight into the factors that regulate proPLT formation and PLP release could be limited since immediate changes to the proPLT formation process cannot be analyzed. Similar limitations in real-time visualizations are present in a porous membrane system through which Mks extended proPLTs into a lower chamber with shear rates of 30-70 s−1.
In contrast, slit bioreactors, which use small features to create <10-μm openings that mimic gaps or fenestrations in endothelial cells lining sinuses in the bone marrow, offer the advantage of in situ study and analysis of proPLT and PLP formation that is difficult in the other types of bioreactors. Some such bioreactors use a 4-μm slit bioreactor with unspecified shear rates and others include a 2-μm slit bioreactor with a shear rate of 500 s−1. Although the PDMS-based fabrication of these slit bioreactors facilitate visualization of the proPLT formation process in real-time, the flow patterns and shear rates within current systems have not been identified. Developing an understanding of the bioreactor flow environment is important since non-uniformity in the flow patterns would lead to Mks experiencing different shear rates depending on slit location.
Embodiments of the present disclosure include a microfluidic proplatelet (proPLT) and platelet-like particle (PLP) production chamber device. In accordance with these embodiments, the device includes a plurality of slit channels that include one or more proPLT/PLP production slits configured to expose a megakaryocyte to a uniform shear profile to facilitate the production of proPLTs/PLPs, with the plurality of slit channels distal to and in fluid communication with a central flow channel.
In some embodiments, the one or more proPLT/PLP production slits are from about 3 μm to about 10 μm wide. In some embodiments, the one or more proPLT/PLP production slits are from about 5 μm to about 7 μm wide. In some embodiments, the one or more proPLT/PLP production slits are about 5 μm wide.
In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits ranging from about 5000 s−1 to about 10,000 s−1. In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits ranging from about 5000 s−1 to about 8,000 s−1. In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits of about 5000 s−1, and the one or more proPLT/PLP production slits are about 7 μm wide. In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits of about 8,200 s−1, and the one or more proPLT/PLP production slits are about 5 μm wide.
In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 50 s−1 to about 500 s−1. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 250 s−1 to about 350 s−1. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 50 s−1 to about 100 s−1. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 250 s−1 to about 350 s−1, and the one or more proPLT/PLP production slits are about 7 μm wide. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 50 s−1 to about 100 s−1, and the one or more proPLT/PLP production slits are about 5 μm wide.
In some embodiments, fluid flows in a single direction from the central flow channel to the plurality of slit channels. In some embodiments, the single direction fluid flow produces at least a 5-fold increase in proPLT/PLP production. In some embodiments, the device further includes a proPLT/PLP collection reservoir.
Embodiments of the present disclosure include a microfluidic bioreactor device for the production of proplatelets (proPLTs) and platelet-like particles (PLPs). In accordance with these embodiments, the device includes a megakaryocyte loading reservoir coupled to a central flow channel, a branching flow channel in fluid communication with the central flow channel, and a proPLT/PLP production chamber that includes a plurality of slit channels distal to and in fluid communication with the branching flow channel, with the plurality of slit channels having one or more proPLT/PLP production slits configured to expose a megakaryocyte to a uniform shear profile to facilitate the production of proPLTs/PLPs.
In some embodiments, the one or more proPLT/PLP production slits are from about 5 μm to about 7 μm wide. In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits of about 5000 s−1, and the one or more proPLT/PLP production slits are about 7 μm wide. In some embodiments, the uniform shear profile produces a maximum shear rate at the one or more proPLT/PLP production slits of about 8,200 s−1, and the one or more proPLT/PLP production slits are about 5 μm wide. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 250 s−1 to about 350 s1, and the one or more proPLT/PLP production slits are about 7 μm wide. In some embodiments, the uniform shear profile produces a shear rate on the proPLTs/PLPs ranging from about 50 s−1 to about 100 s−1, and the one or more proPLT/PLP production slits are about 5 μm wide. In some embodiments, fluid flows in a single direction from the central flow channel to the plurality of slit channels.
Embodiments of the present disclosure include a microfluidic bioreactor system for the production of proplatelets (proPLTs) and platelet-like particles (PLPs). In accordance with these embodiments, the system includes a megakaryocyte loading reservoir coupled to a central flow channel, a branching flow channel in fluid communication with the central flow channel, and a proPLT/PLP production chamber having a plurality of slit channels distal to and in fluid communication with the branching flow channel, with the plurality of slit channels having one or more proPLT/PLP production slits configured to expose a megakaryocyte to a uniform shear profile to facilitate the production of proPLTs/PLPs.
In some embodiments, the system further includes a proPLT/PLP collection reservoir. In some embodiments, the system further includes a fluid source coupled to the central flow channel and configured to supply fluid pressure to the system.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a device” is a reference to one or more devices and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “and/or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and/or C.”
As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The present disclosure relates to the production of platelets from megakaryocytes (Mks). In particular, the present disclosure provides systems and methods for the in vitro production of platelets from Mks using a microfluidic bioreactor having a center flow channel and uniform high-shear micrometer slits. Use of this microfluidic bioreactor enables the continuous production of millions of platelets and facilitates real-time and long-term visualization of proplatelet and platelet generation.
In accordance with the various embodiments of the present disclosure, mature megakaryocytes (Mks) can be continuously introduced into a microfluidic bioreactor using a single flow that distributes itself through smaller branching arms where slits capture the megakaryocytes. The microfluidic bioreactor causes the megakaryocytes to experience shear forces to produce proplatelets and platelets. The microfluidic bioreactor is designed to mimic the lung capillary bed in which branching of larger vessels into smaller channels lead Mks into high shear regions at the slits; hence, it is referred to as “Lung-USRB” (lung uniform-shear rate bioreactor).
Embodiments of the present disclosure involve the application of microfluidic fabrication technology to develop the bioreactor system. In accordance with these embodiments, platelet-like-particles with functional activity have been generated from megakaryocytes introduced into the system. Currently, processed Mk numbers range from about 200K up to about 800K, and are introduced as a single bolus injection. Using an Mk reservoir that continuously supplies a source of Mks can generate millions of Mks over a period of hours.
Embodiments of the present disclosure provide improvements over typical systems and methods for the in vitro production of platelets and proplatelets. In accordance with these embodiments, the present disclosure includes slit bioreactor systems (termed “USRB” for uniform-shear rate bioreactor) that are improvements over previous published slit bioreactors by allowing real-time and long-term visualization of the proPLT formation process within the entire reactor. Embodiments of the USRBs of the present disclosure were filed as U.S. Provisional Application Ser. No. 62/522,491 and U.S. Provisional Patent Application Ser. No. 62/642,955, which are both herein incorporated by reference in their entireties and for all purposes.
Additionally, because the shear rate within currently available slit bioreactor systems was not fully characterized, computational fluid dynamics (CFD) modeling was used to develop the USRBs of the present disclosure and to increase understanding of the platelet formation process. The environment promoted the rapid release of individual PLPs from Mks in the USRBs of the present disclosure, which has been reported in vivo but not in bioreactors in vitro. Initially, the USRBs were designed to mimic the endothelial gaps and fenestrations in the bone marrow, where a primary flow pushes the Mks into slits and a secondary flow exerted shear forces on extending proplatelets, similar to the in vivo process. However, further investigations determined that a single primary flow on the Mks was six-times more efficient at generating proplatelets and platelets and most previously published systems utilized a two-flow set-up. Recent studies have shown that Mks can enter the blood flow and get trapped in the capillary bed of the lung where high shear forces (>2000 s−1) elongate and fragment Mks into platelets within minutes.
Therefore, based on observations of platelet generation in the lung and on data generated using a single-flow system, the USRBs of the present disclosure were modified and scaled up into the “Lung-USRB” system (USRB-5 μm). This novel bioreactor systems of the present disclosure include key features, such as the post geometries, but additionally uses slits with various sizes (e.g., 5-7 μm) to improve capture and to allow flexibility of using Mks derived from different cell sources. For example, as disclosed herein, use of only a center flow channel without the outside flow channels of a USRB-7 μm system, in combination with 5-μm slits of the USRB-5-μm system, led to a surprising and unexpected 6-fold increase in proplatelet production. The USRB-5-μm system with 5-μm slits maintained uniform shear rates with at least a 90-fold increase in capture area, as compared the USRB-7 μm system. Both the USRB-5-μm system and the USRB-7 μm system provide significant advantages over other in vitro systems and methods for platelet generation; however, determining which USRB system to use will depend on various factors, such as particular clinical and/or laboratory needs, as would be recognized by one of ordinary skill in the art based on the present disclosure.
Embodiments of the present disclosure include the use of CFD to guide platelet bioreactor design, analyze forces on Mks, and examine bioreactor performance. In some embodiments, CFD was used to evaluate published slit bioreactors and develop a USRB system design with improved flow and shear uniformity. The 4-μm slit bioreactor introduced by Nakagawa et al. had no specified shear rates. CFD modeling disclosed herein determined rates on proPLTs (along the length of the bioreactor) were within the sinusoids range (200 s−1) and above physiological rates (6,000 s−1), depending on the location. However, the shear rate within the 4-μm slits where Mks would be trapped had a much higher range of 400-30,000 s−1. The substantial nonuniformity of shear rates along the bioreactor length make it difficult to study proPLT/PLP formation real-time since Mks at different regions of the bioreactor would experience substantially different microenvironments.
The 2-μm slit bioreactor developed by Thon et al. had a narrower range of shear rates than Nakagawa et al. Modeling disclosed herein used CFD to analyze this system and determined a shear rate of approximately 500 s−1. CFD outputs showed that, along the length of the bioreactor, proPLTs would experience shear rates from 250 to 500 s−1, and that the slit shear rates ranged from 5,000 to 7,500 s−1. Additionally, Thon et al. provided real-time visualization of the proPLT formation process. However, the shear rates within the slits showed a steady increase along the bioreactor so that Mks trapped at various slits would be exposed to different shear rates.
CFD analysis of the Nakagawa et al. and Thon et al. systems suggests the shear rate across slits increased along the bioreactor from the inlet to the outlet. This can be attributed to the design of the systems in which two parallel-like flows are separated by slits and where the top flow (pushing on the Mks) is redirected into the lower channel at the end of the bioreactor length (Table 1). To avoid generating this increase of shear rates across the slits, USRBs of the present disclosure were instead designed with the two outer flows converged at a 90° C. V-shaped region. This arrangement allowed the center channel flow to push whole-Mk bodies into the 5-7-μm slits with similar maximum shear rates of 5,000 s−1. The outer flow converges at the slits and exerts nearly uniform shear rates (250-350 s−1) on extending proPLTs. Mks exiting the bone marrow sinusoids can be trapped in the vascular bed of the lung where high shear forces are exerted on the whole cell body and on proPLTs, thus, high shear forces were utilized on Mk bodies and physiological shear on the proPLTs. CFD analysis was extended to include cell blockages within the slits and confirmed the flow patterns experimentally to understand behavior of an occupied bioreactor and to estimate anticipated shear rates on proPLTs (100-900 s−1).
Compared to Thon et al., the USRBs of the present disclosure have a similar capture area of 20 slits vs. 15 slits, but a higher slit occupancy (90% vs 66%). More importantly, the slits can be observed during an experimental run and it was noted that on average 40-60% of occupied slits were actively making proPLTs/PLPs. In contrast, the length of the Thon et al. and Nakagawa et al. bioreactors makes it difficult to analyze proPLT/PLP formation from all the slits at the same time. In addition to supporting proPLT production, the USRBs of the present disclosure also promoted rapid release of many individual PLPs, which has not been reported for other published bioreactor systems. This observation is physiologically relevant since Mks have also been observed to make platelets in vivo (in mouse) via a rapid fragmentation process that releases platelets without the proPLT formation step. It was observed that the rate of PLP production appears to be faster when PLPs are rapidly released (Video 1 vs. Video 2). It may be that rapid PLP generation within the USRBs with 5-7 μm slits is largely influenced by the unique slit geometry in which cells are pushed through a hyperbolic-like-converging region. As the area is reduced in this region, whole Mks bodies are squeezed and elongated through the 5-7-μm gap where the shear rate and strain rate are the highest.
Results of the present disclosure show that flow microenvironment can greatly affect the behavior of Mks in real-time. Within the system, it was observed that Mk capture at an open slit increased the release of PLPs across the other Mk-blocked slits. Slit-blockage events could be influenced by the size of the Mks being trapped and are not easily controlled, thus, these observations highlight the importance of understanding how the inherent dynamics of a bioreactor can impact the Mk response. A step-change in the center channel flow rate (while keeping the outside channel flow constant) transiently increased the rate of PLP releases, similarly to the slit-blockage events. The increase in immediate Mk productivity could be attributed to an increase in pressure drop across the slits, as presented by CFD analysis. While a temporary three-fold increase in productivity was observed after cell-blockages or flow rate changes in the USRBs of the present disclosure, it was recognized that the rates are not sustained for the remainder of an experimental run largely due to the dynamic behavior of cell capture and slit openings. Nagakawa et al. did not study the effect of changing the flow conditions within their system. Thon et al. found that the average proPLT extension rate did not change at different flow rates (same flow for both channels, 12.5-100 μL/h), but did not extend their CFD analysis to other flow regimes. Within the USRBs with 5-7 μm slits, it was observed that step-changes to the center channel flow rate or a cell-blockage event led to approximately 30% and approximately 50% increases in active slits, respectively.
It was expected that the presence of an outside channel flow would aid in shearing off proPLTs (increasing the PLP release rate), mimicking physiological blood flow in vivo. Thus, it was unexpected that turning off the outside channel flow rate increased the average number of PLPs released by almost 6-fold. Using CFD simulations, the predicted environment was investigated to try and understand the variables responsible for these unforeseen results. Average velocity profiles, strain rates, and wall shear rates through the slits remained overall unchanged with or without flow in the outer channel. CFD predictions showed a slight increase in back-pressure by the outer flow on the center channel, which could inhibit Mks from releasing more PLPs. However, a significant change in the flow structure was observed, as confirmed by correlations between the CFD streamlines and experimental streamlines. The CFD simulations showed that the slits appear to operate independently from each other with no outside flow (
Analysis of the bioreactor effluent showed CD41+CD42b+ PLP populations that exhibited activation following thrombin addition. Video analysis indicated that approximately 76% of Calcein-stained particles were generated at the slits in reactors with no outside flow. ProPLTs are subjected to a shear environment typical of the bone marrow sinusoids in many of the current published bioreactor systems (
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Embodiments of the USRB systems of the present disclosure include a main or central flow channel entering the USRB that splits into rows that contain left and right arms (branching flow channel). These branching arm channels can have parallel branches that lead to platelet production chambers (proPLTs/PLPs), which include smaller slit channels (
In some embodiments, the layout of the USRB systems of the present disclosure use Murray's law that describes vessel diameters and branching, where the radius of the parent branch (Ro) is related to the sum of the radius of the daughter branches (R1, R2): Ro3=R13+R23. Thus, based on this relationship, a geometry of the USRB systems disclosed was defined as shown in
Embodiments of the present disclosure include microfluidic proplatelet (proPLT) and platelet-like particle (PLP) production chamber devices and systems. In accordance with these embodiments, the device includes a plurality of slit channels comprising one or more proPLT/PLP production slits. In some embodiments, the slits can range in width from about 3.0 μm to about 10 μm, from about 4.0 μm to about 10 μm, from about 5.0 μm to about 10 μm, from about 6.0 μm to about 10 μm, and from about 8.0 μm to about 10 μm. In some embodiments, the slits can range from about 3.0 μm to about 9.0 μm, from about 4.0 μm to about 8.0 μm, and from about 5.0 μm to about 7.0 μm. In some embodiments, the slits can range from about 5.0 μm to about 6.0 μm, including about 5.5-μm.
In some embodiments, the proPLT/PLP production devices and systems of the present disclosure is configured to expose a megakaryocyte to a uniform shear profile to facilitate the production of proPLTs/PLPs. As described herein, uniform shear profiles are based on a number of variables associated with microfluidics in general, and based on certain aspects of the proPLT/PLP production devices of the present disclosure. For example, a uniform shear profile can include a maximum shear rate at a proPLT/PLP production slit. In some embodiments, a uniform shear profile can include a maximum shear rate at one or more proPLT/PLP production slits ranging from about 5000 s−1 to about 10,000 s−1, from about 5,000 s−1 to about 9,000 s−1, from about 5,000 s−1 to about 8,000 s−1, from about 5,000 s−1 to about 7,000 s−1, from about 5,000 s−1 to about 6,000 s−1, from about 5,000 s−1 to about 7,500 s−1, from about 7,000 s−1 to about 10,000 s−1, from about 8,000 s−1 to about 10,000 s−1, or from about 9,000 s−1 to about 10,000 s−1.
In some embodiments, the uniform shear profile includes a maximum shear rate at one or more proPLT/PLP production slits of about 5000 s−1 when the slits are about 7 μm wide. In other embodiments, the uniform shear profile includes a maximum shear rate at one or more proPLT/PLP production slits of about 8,200 s−1 when the slits are about 5 μm wide.
In some embodiments, a uniform shear profile can include a shear rate on proPLTs/PLPs being produced that ranges from about 50 s−1 to about 500 s−1, from about 100 s−1 to about 500 s−1, from about 150 s−1 to about 500 s−1, from about 200 s−1 to about 500 s−1, from about 250 s−1 to about 500 s−1, from about 300 s−1 to about 500 s−1, from about 350 s−1 to about 500 s−1, from about 400 s−1 to about 500 s−1, from about 450 s−1 to about 500 s−1, from about 50 s−1 to about 300 s−1, from about 50 s−1 to about 200 s−1, from about 50 s−1 to about 100 s−1, from about 100 s−1 to about 400 s−1, from about 100 s−1 to about 300 s1, from about 200 s−1 to about 500 s−1, from about 200 s−1 to about 400 s−1, from about 200 s−1 to about 300 s−1, from about 250 s−1 to about 500 s−1, from about 250 s−1 to about 450 s−1, from about 250 s−1 to about 400 s−1, or from about 250 s−1 to about 350 s−1.
In some embodiments, the uniform shear profile includes a shear rate on the proPLTs/PLPs ranging from about 250 s−1 to about 350 s−1 when the one or more proPLT/PLP production slits are about 7 μm wide. In other embodiments, the uniform shear profile includes a shear rate on the proPLTs/PLPs ranging from about 50 s−1 to about 100 s−1 when the one or more proPLT/PLP production slits are about 5 μm wide.
In some embodiments, the proPLT/PLP production devices and systems of the present disclosure include a plurality of slit channels that are distal to and in fluid communication with a central flow channel. In some embodiments, fluid flows in a single direction from the central flow channel to the plurality of slit channels, rather than in a dual flow manner. In accordance with these embodiments, a single direction fluid flow can produce at least a 5-fold increase in proPLT/PLP production, and in some cases, at least a 6-fold increase in proPLT/PLP production.
In some embodiments, the proPLT/PLP production devices and systems of the present disclosure include a proPLT/PLP collection reservoir into which proPLTs/PLPs can be collected after formation. The proPLT/PLP collection reservoir is generally the most distal component of the present devices and systems, and in some cases, more than proPLT/PLP collection reservoir can be used. In some embodiments, the proPLT/PLP production devices and systems of the present disclosure include a megakaryocyte loading reservoir coupled to a central flow channel, and in some cases, megakaryocyte loading reservoir is the most proximal component of the present devices and systems. As described herein, a plurality of branching flow channels are located between the megakaryocyte loading reservoir and the proPLT/PLP collection reservoir. In accordance with these embodiment, the present devices and systems can also include a fluid source coupled to the central flow channel and configured to supply fluid pressure to the system to facilitate proPLT/PLP production.
Embodiments of the present disclosure also include methods for producing proPLTs/PLPs using the devices and systems described above, as would be recognized by one of ordinary skill in the art.
Embodiments of the present disclosure also include methods of manufacturing a USRB device and/or system, as well as a proPLT/PLP production chamber. Material and methods for manufacturing or fabricating the proPLT/PLP production devices and systems of the present disclosure are generally known to one of ordinary skill in the art. For example, a 2D design of the bioreactor was created in AutoCAD 2014 and then printed onto a chrome mask. A silicon wafer was spin-coated with SU8-2035 photoresist at 4000 RPM for 30 s to achieve a photoresist height of 40 μm. The wafer was soft-baked at 65° C. for 3 min and then hard-baked at 95° C. for 6 min. Afterwards, the wafer was exposed to UV light, using a Karl Suss MA6 Mask Aligner, for 17 s. The exposed resist was placed at 95° C. for 6 min (post-exposure bake).
The resist was developed using SU8 developer solution for 2 min and dried with a nitrogen gun. The dry wafer was silanized overnight in a vacuum chamber. Next, a 1:10 curing agent to polydimethyl siloxane (PDMS) solution was poured over the wafer to cast a mold that was placed in an oven at 65° C. overnight. The PDMS mold was then cut, holes for inlets and outlets punched with a 2-mm punch, and the PDMS plasma-bonded to an ethanol-cleaned premium plain glass slide.
As one of ordinary skill in the art would recognize based on the present disclosure, other materials and methods may be used to manufacture or fabricate one or more components of the proPLT/PLP production devices and systems of the present disclosure.
CFD Modeling. Flow simulations were conducted using ANSYS v16.1 (ANSYS, Inc., Canonsburg, Pa.). 3D models of the slit bioreactors were created using Autodesk Inventor Professional software 2015 (San Rafael, Calif.) and the files imported into ANSYS Design Modeler. A computational grid (mesh) was individually optimized for each system (
A summary of platelet slit bioreactors published to date is provided in Table 1. A description of the system and operation, as well as key dimensions, is given for each bioreactor. CFD modeling is presented below for the slit-type bioreactors.
Methodology. The slit bioreactor simulations were carried out using ANSYS version 16.1 (Canonsburg, Pa.) that includes the computational fluid dynamics solver FLUENT. The 3D models were created in Autodesk Inventor Professional software 2015 (San Rafael, Calif.). The files were converted to Parasolid binary text in Inventor and then imported into ANSYS Design Modeler. A mesh was then created for each system, in which the geometry is discretized into small volumes (elements) where the CFD calculates an approximate solution to the discretized form of the governing equations. The mesh for each individual system was optimized to yield a converging solution that is mesh-independent. The acceptance criteria used was a change in the predicted CFD velocity of no more than 5% from the previous converged solution. Boundary conditions were no-slip at the walls, constant inlet velocity, and default gauge pressure of 0 Pa at the outlet. The velocity input into each system was determined from the volumetric flow rate and the dimensions specified by the authors. FLUENT was used to solve the steady-state form of the Navier-Stokes Equation (Eq. 1) for an incompressible Newtonian fluid subjected to the specified flow conditions. The convergence tolerance for all simulations, which is the normalized residual for each degree of freedom, was set to 10−3. The overall methodology is shown in
Overall, the flow rates and designs lead to laminar flow conditions inside the bioreactors, such that viscous forces dominate inertial forces. Thus, the viscosity of the media used for perfusion through the systems and the operating temperature have a substantial impact on the expected shear forces within the bioreactors. Table 2 shows viscosity measurements for different media at 37° C. The measured fluid viscosity was used in the simulations.
All simulations for the systems were run with the following computer and software settings. Computer Specs: Dell Precision T1700, Intel® Core™ i7-4790 CPU @ 3.60 GHz, 32 GB RAM, 64-bit, Windows 10 Pro.
FLUENT Settings:
Modeling of Slit Bioreactors—Nakagawa et al. First, the minimum element size was specified for the entire geometry. Next, the geometry was subdivided into two parts: Body and Slits. Using this approach, the 4-μm slits could be assigned a specific element size that could sufficiently resolve these small regions. The top and bottom chambers (Body) were assigned identical element sizes. The element size for the slits matched the minimum element size for the entire geometry. The mesh was further refined by reducing the slit's element size as part of the mesh-independence study. This approach avoids creating an excessive number of elements in the body region that are not needed, while resolving additional detail in the slit region. The final mesh-independent system and mesh settings are shown in
This bioreactor system contains a primary flow that pushes Mks into the 4-μm slits and a second flow that shears off proPLTs from Mks. Both flow rates are 16.7 μL/min, which corresponds to an inlet velocity of 5.85 cm/s. Velocity analysis of the Nakagawa et al. system focused on the flow patterns in the slits as well as the net flow along the length of the reactor (
Modeling of Slit Bioreactors—Thon et al. Similar to the approach mentioned for Nakagawa et al., the distribution of the elements in the computational mesh for the Thon et al. system was refined in the region of the slits and coarser in the parallel flow channels. The mesh was further refined by sequentially reducing the element size in the slits. The final mesh-independent system and mesh settings are shown in
Modeling of Slit Bioreactors—Uniform-shear rate bioreactor (USRB-7 μm). Initially, the whole system, as shown in
The bioreactor system contains a primary flow down the center channel that pushes Mks into the 7-μm slits and an outer channel flow that shears off proPLTs from Mks. The center channel flow rate and the combined outer channel flow rate are both 1.5 μL/min. Since the geometry was cropped, the input velocity of the center channel is set to 0.0694 cm/s (V1 in
PLP Quantification. Platelet-like-Particles (PLPs) were counted on proPLTs based on the number of “beads” observed (
Bioreactor Fabrication. Briefly, a chrome mask (Front Range Photomask, Palmer Lake, Colo.) of the USRB-7 μm was used to create a master silicon wafer (WRS Materials, San Jose, Calif.) on which polydimethyl siloxane (PDMS) solution (Slygard 184 Kit; Electron Microscopy Sciences, Hatfield, Pa.) was poured to cast a mold.
A 2D design of the bioreactor was created in AutoCAD 2014 (San Rafael, Calif.) and then printed onto a chrome mask (Front Range Photomask, Palmer Lake, Colo.). A silicon wafer (WRS Materials, San Jose, Calif.) was spin-coated with SU8-2035 photoresist (MicroChem Corp, Westborough, Mass.) at 4000 RPM for 30 s to achieve a photoresist height of 40 μm. The wafer was soft-baked at 65° C. for 3 min and then hard-baked at 95° C. for 6 min. Afterwards, the wafer was exposed to UV light for 17 s using a Karl Suss MA6 Mask Aligner (SUSS MicroTec, Garching, Germany). The exposed resist was then baked at 95° C. for 6 min. Finally, the resist was developed using SU8 developer solution (MicroChem) for 2 min and dried with a nitrogen gun. The dry wafer was silanized overnight (5 μL of 1H,1H,2H,2H-perfluorooctyltrichlorosilane; Alfa Aesar, Ward Hill, Mass.) in a vacuum chamber. Next, a 1:10 curing agent to polydimethyl siloxane (PDMS) solution (Slygard 184 Kit, Electron Microscopy Sciences, Hatfield, Pa.) was poured over the wafer to cast a mold that was placed in an oven at 65° C. overnight. The PDMS mold was then cut, holes for inlets and outlets created with a 2-mm punch, and the PDMS plasma-bonded (Model BD-20; Electro-Technic Products, INC, Chicago, Ill.) to an ethanol-cleaned premium plain glass slide (25×75×1 mm; VWR, Radnor, Pa.).
Cell Culture. Unless otherwise specified, all reagents were obtained from Sigma-Aldrich (St. Louis, Mo.), and cytokines from Peprotech (Rocky Hill, N.J.). Media viscosity used in the CFD simulations was measured using a Cannon-Fenkse Routine Viscometer—size 50 (Cannon Instrument Co., State College, Pa.). Two measurements were conducted for IMDM, IMDM+10% FBS, and IMDM+20% BIT, at 37° C. in a water bath. Previously frozen mobilized peripheral blood (mPB) CD34+ cells from the Fred Hutchinson Cancer Research Center (Seattle, Wash.) with Northwestern University Institutional Review Board approval were grown in 78% IMDM (Gibco, Carlsbad, Calif.), 20% BIT 9500 Serum Substitute (STEMCELL, Vancouver, BC, Canada), 1% Glutamax (Gibco), 1 μg/mL low-density lipoproteins (Calbiochem, Whitehouse Station, N.J.), 100 U/mL Pen/Strep, 100 ng/mL TPO, 100 ng/mL SCF, 10 ng/mL IL-6, 10 ng/mL IL-11, and 2.5 ng/mL IL-3 (R&D Systems, Inc., Minneapolis, Minn.). Cells were maintained between 100,000 to 400,000 cells/mL at 37° C., 5% CO2, and 5% O2 for 5 days (Panasonic incubator MCO-170M, Wood Dale, Ill.). On day 5, the cytokines were replaced with 100 ng/mL TPO, 100 ng/mL SCF, 10 ng/mL IL-9, 10 ng/mL IL-11, and 10 ng/mL IL-3. Cells were maintained at a density of 250,000 to 500,000 cells/mL and kept at 37° C., 5% CO2, and 20% O2 until day 7. On day 7, cells were selected using anti-CD61-conjugated magnetic microbeads (Miltenyi Biotech Inc, San Diego, Calif.) and then cultured in medium with 100 ng/mL TPO, 100 ng/mL SCF and 6.25 mM nicotinamide thereafter. The cells were maintained at a density between 250,000 to 500,000 cell s/mL and kept at 37° C., 5% CO2, and 20% O2.
Bioreactor Perfusion with Mks. The USRB-7 μm was positioned on a Lumascope microscope v500 (Etaluma Inc., Carlsbad, Calif.) placed inside an incubator (Thermo Scientific, Waltham, Mass.) maintained at 37° C. and 5% CO2. Separate syringe pumps (NE-300, New Era. Pump Systems Inc., Farmingdale, N.Y.) were used for each flow channel. A 5-mL glass syringe (81520, Hamilton Company, Reno, Nev.) was used for the outer channels and a 2.5-mL glass syringe (81420, Hamilton) was used for the center channel. Media (78% IMDM (Gibco, Carlsbad, Calif.), 20% BIT 9500 Serum Substitute (STEMCELL, Vancouver, BC, Canada), 1% Glutamax (Gibco), 1 mg/mL low-density lipoproteins (Calbiochem, Whitehouse Station, N.J.), 100 U/mL Pen/Strep) without cytokines was perfused throughout the bioreactor for 30 min at 6.5 μL/min prior to Mk introduction. On day 10, 11, or 12 of Mk culture, Mks at density of 50,000/mL were stained for 15 min with 1 μM Calcein AM at 37° C. After the 30-min media perfusion, 25,000 Mks (a sufficient number to observe the system dynamics and how often they might repeat and under what conditions, without clogging the slits) were microinjected into the tubing upstream from the reactor. No Mks were present within the syringes. A video was recorded of each bioreactor for 1-2 h.
Video Analysis. Videos (6 frames-per-second) were recorded for each experimental run using the Lumascope v500, equipped with High Sensitivity Monochrome CMOS Sensor camera, using a 20× or 40× objective. Each video was analyzed for every 5-min time interval for the duration of an experiment. One half of the bioreactor (10 slits) was analyzed at one time throughout 5-min time intervals for the entire video recorded. This process was repeated on the other half of the reactor. The data from each half of the reactor was then combined for each 5-min time interval. During each interval, only proPLTs and PLPs that originated from trapped. Mks in the slits were counted. Additionally, for some videos, pre-released particles flowing into and out of the slits were counted separately. Mks can give rise to particles without shear and these could be present in the suspension that was microinjected into the system. To increase accuracy, the videos were played at a slower speed during times of high PLP release activity. The 5-min interval was selected because it allowed effective analysis and perturbation of the dynamics of the process. Pre-staining Mks with Calcein AM allowed the Mks trapped in the reactor, as well as proPLTs/PLPs, to be clearly observed.
The following videos are part of the present disclosure and are available upon request:
Video 1: proPLT Formation. Trapped Mk extending proPLTs through slit. Flow in the outer channels applies shear on the extensions further elongating them leading to fragmentation after several minutes. Time units: h:min:s. Scale bar=50 μm. Flow rate of 1.5 μL/min in the center channel with 5 μL/min in the combined outer channels.
Video 2: Rapid PLPs releases. Trapped Mks in a slit, rapidly releasing many individual PLPs in seconds. Time units: h:min:s. Scale bar=35 μm. Flow rate of 1.5 μL/min in the center channel with 5 μL/min in the combined outer channels.
Video 3: Major slit-blockage by Mks. Upon cell-capture at a slit, there is a noticeable increase in proPLT/PLP activity across the bioreactor, Scale bar=50 μm. Flow rate of 1.5 μL/min in the center channel with 5 μL/min in the combined outer channels.
Video 4: Impact of turning off outside flow rate. Starting with an outside flow rate at 5 μL/min, Mks observed making proPLTs/PLPs. After turning off the outside flow rate, there is an increase in proPLT/PLP productivity across the bioreactor. Scale bar=50 μm. Flow rate of 1.5 μL/min in the center channel.
Video 5: Rapid proPLT formation and PLP releases. Mks are trapped and rapidly converted to proPLTs/PLPs at the slits. Scale bar=50 μm. Flow rate of 1.5 μL/min in the center channel with 0 μL/min in the combined outer channels.
Statistical Analysis. JMP Pro 11 (SAS Institute Inc., Cary, N.C.) was used to generate histograms, distributions, and standard errors of the video analysis data for released proPLTs/PLPs.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
Computation fluid dynamics (CFD) was used to evaluate the flow and shear conditions within several slit bioreactors (Table 1), CFD analysis of the Nakagawa bioreactor (
A thorough CFD analysis was conducted on potential new slit bioreactor designs to avoid the CFD-predicted nonuniform flow and shear profiles of current slit bioreactors. In the optimized bioreactor system of the present disclosure, Mks enter a center channel where a V-shaped array of twenty 7-μm slits separates the Mks from outside flows converging at 90° C. (
Cell-blockage scenarios for the slits were simulated using CFD, with a center channel flow rate of 1.5 μL/min and a combined outer channel flow rate of 5 μL/min. First, 20-μm sized spheres were modeled just upstream of slits (i.e., partial blockage). The simulations predict that these cells would experience a shear rate of 1,500-3,000 s−1 (
After design and fabrication of the USRB-7 μm (
Example 5
The kinetics of the USRB-7 μm were analyzed to identify conditions that change Mk behavior by counting the number of Calcein-stained PLPs that originate from Mks trapped at the slits per 5-min time interval across individual experimental runs (
While trying to remove a small bubble from the outer channel during an experiment, the flow of the outer channels was inadvertently stopped. Surprisingly, when the outer channel flow was completely stopped, the rate of proPLT and PLP release dramatically increased (
Bioreactor runs were compared using outer channel combined flow rates of 5 μL/min (9 bioreactor runs across 3 different Mk cultures) and 0 μL/min (4 bioreactor runs across 2 different Mk cultures) (
It is important to demonstrate that the PLPs produced exhibit functional activity. Due to the higher productivity, the effluent of three bioreactors operated with an outer channel combined flow rate of 0 μL/min was analyzed. Calcein+ PLPs were approximately 67% CD41+CD42b+ (
The effluent most likely contained a combination of pre-released particles (present in the Mk suspension introduced into the system) and PLPs generated at the slits. The videos using the counting strategy described earlier were analyzed to determine the rate at which pre-released Calcein-stained particles entered and exited the slits. The mean rate was 125 per 5-min time interval (
To verify the PLP counting process, expired blood platelets, stained with Calcein, were introduced into a cell-free bioreactor. During a 30-min perfusion, platelets were counted per 5-min time interval for outer channel combined flow rates of 5 μL/min or 0 μL/min (
CFD was used to evaluate what environmental factors could explain the differences in Mk behavior at 5 μL/min vs. 0 μL/min flow rates in the outer channels, while keeping the center channel flow rate constant at 1.5 μL/min. Wall shear rate, pressure, velocity, strain rate, and the structure of the flow patterns were the primary factors of interest. Simulations with the outer flow rate of 0 μL/min did not show changes to the wall shear rates within the slits of the bioreactor (
Next, the strain rate (rate of deformation) within the bioreactor slits was examined. Strain rates represent extensional flow that is created due to a velocity gradient in the direction of flow. The CFD outputs of the bioreactor of the present disclosure showed an increase in velocity along the slits, due to the hyperbolic like-converging region (
The structure of the flow patterns was assessed using the CFD streamlines, as well as 1-μm fluorescent beads to map the experimental streamlines. There is significant correlation between the predicted and experimental streamlines under the two different outside flow conditions (
Previous studies have investigated platelet generation from mobilized-peripheral blood (mPB) Mks (e.g., Martinez, A F et al. Biotechnology Progress 2017; 33:1614-1629). Mks can also be derived from cord-blood stem and progenitor cells (CB). These CB-Mks are known to be lower ploidy and smaller in size compared to mPB-Mks. In some embodiments, the USRB-7 μm with CB-Mks showed that though cells can get captured, the overall efficiency was lower due to smaller size as these cells more easily squeezed through the slits. Images depicting the USRB-7 μm loaded with CB-Mks are shown in
To improve the retention of CB-Mks at the slits, the slits were narrowed to 5 μm and termed “USRB-5 μm,” which retains the same slit geometry and design as the USRB-7 μm described above. This system exposed Mks to a uniform environment with max shear rate at the slits of 8200 s'1 and shear on proPLTs at 50-90 s−1 (
The Mk bone marrow niche is a complicated environment to replicate. Besides cell-cell and cell-extracellular matrix interactions, several abundant proteins exist within this environment, of which fibrinogen and fibronectin had some observed effect on platelet production, among others. Both of these proteins have a positive impact on in vitro proplatelet formation. Due to the design and materials of the USRB-7 μm and USRB-5 μm bioreactors, coatings were introduced into the system to then evaluate Mk-protein interaction in the presence of uniform shear forces to potentially elucidate additional key effects on platelet production. Using a fluorophore-tagged fibrinogen protein, results showed that the USRB systems were uniformly coated across the slits (
The data presented herein show how the USRB systems can be used to characterize Mk behavior within well-defined microfluidic environments. Changing the slit size to allow better capture facilitated more efficient investigations of CB-Mks. Additionally, incorporating other aspects of the bone marrow microenvironment into the USRBs will allow further analysis platelet generation from Mks.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/522,491 filed Jun. 20, 2017, and U.S. Provisional Patent Application No. 62/642,955 filed Mar. 14, 2018, both of which are incorporated herein by reference in their entireties for all purposes.
This subject matter of this invention was made with Government support under Grant No. 5R01HL130760-03 awarded by the National Institutes of Health (NHLBI), and Grant No. CBET1265029 awarded by the National Science Foundation (NSF). The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/038579 | 6/20/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62642955 | Mar 2018 | US | |
| 62522491 | Jun 2017 | US |
