Patent Application
20230132012
The invention relates generally to microfluidic systems, and more particularly to rotary via valves, microbioreactors, and applications of the same.
The background description provided herein is for the purpose of generally presenting the context of the invention. The subject matter discussed in the background of the invention section should not be assumed to be prior art merely as a result of its mention in the background of the invention section. Similarly, a problem mentioned in the background of the invention section or associated with the subject matter of the background of the invention section should not be assumed to have been previously recognized in the prior art. The subject matter in the background of the invention section merely represents different approaches, which in and of themselves may also be inventions. Work of the presently named inventors, to the extent it is described in the background of the invention section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the invention.
The need to study the mechanisms of infection and response to therapies of highly infectious pathogens, such as the eastern, western, and Venezuelan equine encephalitis alphaviruses, and the highly infective coronaviruses, such as SARS-CoV, MERS-CoV, and SARS-CoV-2, require the use of biosafety level 3 (BSL-3) or BSL-4 facilities to ensure containment of the pathogen and protection of the researchers studying them. Conventional two-dimensional (2D) monoculture of cells on the bottom of a plastic flask, Petri dish, or well plate is inadequate to recapitulate the complexity and multi-organ involvement of the diseases associated with these pathogens. For example, the Venezuelan equine encephalitis alphavirus (VEEV) might enter the brain either through the olfactory bulb via the nasal cavities, or by entering the circulation at the lungs and then crossing the blood-brain barrier. Infection with SARS-CoV-2 can result in pulmonary, central nervous system (CNS), and gastrointestinal (GI) involvement in the initial stages of the infection, a massive inflammatory response, and multi-organ failure, and the detailed sequence of events during this coupled infection/inflammatory response is poorly understood. Microphysiological systems (MPS) models that utilize individual or coupled 2D or 3D organs-on-chips or tissue chips have been shown to better recapitulate human physiology and pathology than 2D cultures on plastic, but the existing devices are ill-suited for use in BSL-3 and BSL-4 facilities, which require strict secondary containment of all experiments involving pathogens and thorough decontamination by solvent immersion, autoclaving, or other techniques of hardware or samples being removed from the BSL-3 or BSL-4 facilities. The easiest way to decontaminate disposable materials and supplies coming out of such facilities is by incineration. Hence the maintenance and repair of complex instruments within a BSL-3 or BSL-4 environment presents major technical and economic challenges.
Living cells require fresh media to provide nutrients and remove toxic waste products. In the human body, nutrients are supplied through the GI tract, and metabolites are detoxified or eliminated by the kidney, liver, gut, lungs, skin, and other organs. For in vitro studies, 2D culture on plastic and MPS are distinguished primarily by the means for media exchange: in 2D culture a substantial fraction of the media is exchanged every day or two, thereby depriving the cells of the signaling and metabolic factors that condition the media. In MPS models, continuous or repetitive perfusion provides the fresh media and removes metabolic waste products. The perfusion flow rate and volumes can be adjusted to minimize the dilution of signaling molecules and metabolites and wash away signaling factors and metabolites that should be at a low concentration in normal tissue. Hence perfusion maintains these concentrations at the appropriate levels for both physiological effect and detectability.
Many commercial and research MPS models offer impressive capabilities but require a high level of both infrastructure and personnel expertise. Some commercial MPS lung-on-a-chip, airway chip, and liver-chip assay platforms include perfusion control hardware that is large, interconnected by tubing to pressurize reservoirs pneumatically or for peristaltic pumping to perfuse the chips that cannot be easily utilized in a BSL-3 lab, expensive, and not disposable. Other platforms require external syringe pumps and specially trained personnel to run their devices, as well as pumps with pressure fit connections that could pose a leak hazard in the BSL-3 environment. Systems with tubing and pumps often require bubble traps, since small changes in the temperature of the media being pumped can lead to the release of bubbles which, having no other means of escape, can strip cells from the surfaces of bioreactors as the fluid is pumped through tubing, channels, and chambers. Tissue chip systems that use rockers for gravity perfusion are ill-suited for BSL-3 and often cannot adequately recapitulate the continuous, unidirectional flow of the in vivo environment. In the current COVID-19 crisis and to meet a high demand for effective research tools that are BSL-3 compliant, there is a clear need for an MPS chip that is both easy to make and easy to use, and that provides physiologically relevant responses to viral and pharmaceutical challenges.
Often gravity-induced flow through a microfluidic network or a bioreactor is a practical and very inexpensive alternative to using complicated and bulky pumps. For applications that do not require fast flows or repeated adjustments of the flow rate, or where the use of external pumping devices is cost or space prohibitive, gravity-induced flow offers a simple solution. The chip can contain both the supply and collection reservoirs and be tilted to provide the height difference between the two; without tilting, open, vertical pipette tips can be inserted into the fluidic channels, or, also without tilting, cylindrical glass reservoirs can be bonded to the chip to deliver fluid and store waste, with the flow rate depending upon the height difference between the supply and waste reservoirs.
Gravity perfusion has been successfully used in the past for long-term cell culture in a thick tissue bioreactor (TTB). A simple way to control the flow rate in the gravity driven system for a given pressure differential without tilting is to have control over fluidic resistance of the network. This approach was implemented in our TTB by introducing a constricting output manifold. By selecting a manifold with an appropriate channel height, one can produce a sufficient resistance to flow that, in combination with the fixed height of the supply reservoir, could be used to passively limit the flow through the bioreactor. Such an approach works well for a given fluidic configuration but lacks flexibility. An adjustable valve that can change fluidic resistance of the flow path depending on experimental requirements would be a significant improvement and a more universal solution to the passive flow regulation problem.
Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies.
One of the objectives of this invention is to provide a new class of gravity or pressure-perfused bioreactors that are designed specifically for use in a BSL-3 or BSL-4 environment, but have a large number of other possible applications in drug discovery, toxicology, biology, and medicine, to meet the above-noted needs.
In one aspect, the invention relates to a vertical-via rotary valve (VVRV), comprising: a valve body having a housing; one or more fluidic channels, each fluidic channel having a vertical channel portion defined in the valve body and being adjacent to the housing, wherein a fluid flow through the vertical channel portion is controllable by deforming a sidewall of the vertical channel portion; and an actuator received in the housing and rotatably engaged with the one or more fluidic channels to operably control the fluid flow through the vertical channel portion of each fluid channel.
In one embodiment, the valve body is formed of an elastomeric material including polydimethylsiloxane (PDMS).
In one embodiment, the vertical channel portion of each fluidic channel is a vertical via.
In one embodiment, the actuator features a vertical trough aligned with the vertical via, such that when the actuator un-pinches the vertical via and the fluid flow through the vertical via is allowed, and when the actuator rotates from the un-pinched position, the vertical via is pinched closed and the fluid flow is restricted.
In one embodiment, the vertical channel portions of the one or more fluidic channels in the valve body are formed in a single layer or multiple layers in the valve body.
In one embodiment, the vertical channel portion of each fluidic channel is in fluid communication with a first port and a second port.
In one embodiment, the actuator comprises one or more rolling members, and a driving member for driving the one or more rolling members to rotate independently and/or coordinately, such that when actuated, the one or more rolling members selectively address the vertical channel portions of the one or more fluidic channels so as to control rates of the fluid flow through the vertical channel portions of the one or more fluidic channels independently and/or coordinately.
In one embodiment, the one or more rolling members comprise one or more actuating discs, wherein each actuating disc is formed with a tapered or raised protrusion such that rotation of said actuating disc results in the vertical channel portion of a corresponding fluidic channel in a fully closed state, a partially closed state, or a fully open state, depending upon a position of said tapered or raised protrusion relative to the vertical channel portion.
In one embodiment, the one or more actuating discs are concentrically stacked to one another.
In one embodiment, the driving member comprises one or more nesting wrenches.
In one embodiment, the one or more rolling members comprise a plurality of rollers or a plurality of planetary gears rotatably engaged with the driving member.
In one embodiment, the driving member comprises a top plate, a bottom plate and a central hub connected to central portions of the top plate and the bottom plate, and wherein the plurality of rollers is rotatably affixed to edge portions of the top plate and the bottom plate, such that rotation of the central hub rotates causes each roller to rotate around its own axis but not translate relative to the top plate and the bottom plate.
In one embodiment, the driving member comprises a top plate, a bottom plate, a driving shaft connected to central portions of the top plate and the bottom plate, and a sun gear affixed to the driving shaft and situated between the top plate and the bottom plate, wherein each planetary gear is meshed with the sun gear and rotatably affixed between the top plate and the bottom plate, such that the rotation of the drive shaft is transferred to the sun gear, which in turn causes each planetary gear to rotate around its own axis but not translate relative to the top plate and the bottom plate.
In one embodiment, the actuator comprises: one or more actuating pins accommodated in a pin housing and corresponding to the vertical channel portions of the one or more fluidic channels, wherein each actuating pin is movable from a first position in the pin housing to a second position out of the pin housing when pushed, otherwise, said actuating pin returns from the second position to the first position, wherein when said actuating pin is in the first position, it un-pinches a corresponding vertical channel portion so that the fluid flow therethrough is not restricted, and when said actuating pin is in the second position, it pinches the corresponding vertical channel portion so that the fluid flow therethrough is restricted; a pin actuator cam having at least one lobe; and a driving shaft engaged with the pin actuator cam to operably rotate the pin actuator cam, such that when the lobe is aligned with an actuating pin, the lobe pushes said actuating pin to move from the first position to the second positon, thereby restricting the fluid flow through said corresponding vertical channel portion, when the lobe is misaligned with said actuating pin, said actuating pin returns from the second position to the first position, thereby, unrestricting the fluid flow through said corresponding vertical channel portion.
In one embodiment, each actuating pin is accommodated in the pin housing with an elastic member including a spring.
In one embodiment, the return force on the actuator pin is provided by the elastomeric material of the valve body, including PDMS.
In one embodiment, the VVRV is a mixing valve.
In one embodiment, the valve body is disposable, while the actuator is never in contact with fluid and is reusable.
In another aspect, the invention relates to a gravity perfused bioreactor, comprising: a plurality of chambers vertically stacked to one another on a substrate; and a plurality of reservoirs disposed over the plurality of chambers, wherein each chamber has an input port and an output port fluidically coupled to two corresponding reservoirs of the plurality of reservoirs, respectively; and wherein the plurality of reservoirs and the plurality of chambers are configured such that fluid flows between the plurality of reservoirs and the plurality of chambers are gravity-fed.
In one embodiment, the gravity perfused bioreactor is devoid of a valve.
In one embodiment, the gravity perfused bioreactor further comprises a plurality of permeable membranes, wherein each permeable membrane is disposed between two vertically adjacent chambers.
In one embodiment, the plurality of chambers comprises an endothelial chamber and an epithelial chamber.
In yet another aspect, the invention relates to a gravity perfused bioreactor, comprising: a ribbon fluidics comprising supply channels, collection channels, and a fluid manifold fluidically coupled between the supply channels the collection channels; a bioreactor disk attached to the ribbon fluidics, comprising a bioreactor fluidically coupled to the fluid manifold of the ribbon fluidics; a plurality of supply reservoirs fluidically coupled to the supply channels of the ribbon fluidics; and a plurality of collection reservoirs fluidically coupled to the collection channels of the ribbon fluidics, wherein the plurality of supply and collection reservoirs and the bioreactor are configured such that fluid flows between the plurality of supply and collection reservoirs and the bioreactor are gravity-fed.
In one embodiment, the gravity perfused bioreactor is devoid of a valve.
In one embodiment, each reservoir comprises an intubation recess.
In one embodiment, the ribbon fluidics comprises an inverted, twisted ribbon fluidics.
In one embodiment, the ribbon fluidics is adapted for invertible loading.
In one embodiment, the supply reservoirs, the bioreactor, and the collection reservoirs are terraced for passive pumping.
In one embodiment, the gravity perfused bioreactor further comprises an openable puck housing and an openable puck compression ring coupled to the bioreactor.
In one embodiment, the bioreactor is mounted on a microscope slide.
In one embodiment, the supply reservoirs are situated at a higher elevation than the collection reservoirs.
In one embodiment, the bioreactor comprises a plurality of bioreactor layers vertically stacked to one another, which in turn is detachably attached to the fluid manifold.
In one embodiment, the gravity perfused bioreactor further comprises a plurality of vertical-via rotary valves (VVRVs), wherein each VVRV is fluidically coupled between one of the supply reservoirs and the collection reservoirs and the bioreactor.
In one embodiment, the gravity perfused bioreactor further comprises at least one pump for inducing the fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers.
In one embodiment, the gravity perfused bioreactor further comprises external pressure or suction sources including mechanical pumps or external gravity-feed/collection reservoirs.
In one embodiment, the flow rates and pressures are adjustable on-the-fly by changing the relative pressure differential between the supply and collection reservoirs by adjusting the relative elevation of supply fluid of the supply reservoirs to collection fluid of the collection reservoirs.
In one embodiment, the flow rates and pressures are adjustable by changing perfusate volume in the reservoirs or elevating the supply reservoirs relative to the collection reservoirs.
In a further aspect, the invention relates to a gravity perfused bioreactor, comprising: a plurality of bioreactor chambers stacked to one another, wherein each chamber has an input port and an output port; a plurality of reservoirs disposed over the plurality of bioreactor chambers; and a plurality of vertical-via rotary valves (VVRVs), wherein each VVRV is disclosed above and fluidically coupled between a corresponding reservoirs of the plurality of reservoirs and one of the input port and the output port of a corresponding chamber of the plurality of bioreactor chambers.
In one embodiment, the gravity perfused bioreactor further comprises a plurality of permeable membranes, wherein each permeable membrane is disposed between two adjacent bioreactor chambers.
In one embodiment, fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers are gravity-fed, and/or induced by mechanical pumping.
In one embodiment, the gravity perfused bioreactor further comprises at least one pump for inducing the fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers.
In one embodiment, the gravity perfused bioreactor further comprises external pressure or suction sources including mechanical pumps or external gravity-feed/collection reservoirs.
In one aspect, the invention relates to a gravity perfused bioreactor system, comprising: at least one bioreactor chamber; and a plurality of reservoirs, wherein the plurality of reservoirs comprises an autofilling supply reservoir, a bioreactor supply reservoir, and a bioreactor collection reservoir; wherein the at least one bioreactor chamber is fluidically coupled between the bioreactor supply reservoir and the bioreactor collection reservoir; and wherein the autofilling supply reservoir is fluidically coupled to the bioreactor supply reservoir by a reservoir delivery tube and a level-sensing air vent and configured with adjustable level control for a constant pressure head, such that media in the autofilling supply reservoir is released only when a fluid level in the bioreactor supply reservoir drops below the lower end of the level-sensing air vent, at which point air enters the autofilling supply reservoir, increases a volume of air trapped in the autofilling supply reservoir, and allows fluid to move from the autofilling supply reservoir to the bioreactor supply reservoir through the reservoir delivery tube.
In one embodiment, the media from the bioreactor supply reservoir then flows through the at least one bioreactor chamber into the bioreactor collection reservoir, thereby increasing the level of media in the bioreactor collection reservoir.
In one embodiment, the gravity perfused bioreactor system further comprises a vertical adjustment means coupled to the autofilling supply reservoir for setting the fluid height in the bioreactor supply reservoir.
In one embodiment, the autofilling supply reservoir is situated over the bioreactor supply reservoir, or located remote from the bioreactor supply reservoir.
In one embodiment, the gravity perfused bioreactor system further comprises a wick fluidically coupled to the bioreactor collection reservoir for adjusting the fluid level in the bioreactor collection reservoir.
In one embodiment, the wick comprises a collection bag, a collection tube having one end placed in the bioreactor collection reservoir and the other end placed in the collection bag, and a collection wick received in the collection tube.
In one embodiment, the gravity perfused bioreactor system further comprises a siphon tube inserted in a side punched hole of the bioreactor collection reservoir for adjusting the fluid level in the bioreactor collection reservoir.
In one embodiment, the autofilling supply reservoir is a large-volume airtight reservoir that maintains a constant supply reservoir height therein, and the wick or tube collection system ensures that the collection fluid in the bioreactor collection reservoir has a near-constant height so that media flows from the airtight autofilling reservoir, through the bioreactor supply reservoir, and perfuses the bioreactor at a constant flow rate.
In another aspect, the invention relates to a gravity perfused bioreactor system, comprising: a first reservoir and a second reservoir; at least one bioreactor chamber having at least one first channel fluidically coupled to the first reservoir, and at least one second channel fluidically coupled to the second reservoir; and a valve comprising: at least one first valve pin situated on the at least one first channel between the first reservoirs and the at least one bioreactor chamber, such that when the at least one first valve pin is pushed, fluid flow through the at least one first channel is occluded, and when the at least one first valve pin is released, the fluid flow through the at least one first channel is allowed; at least one second valve pin situated on the at least one second channel between the at least one bioreactor chamber and the second reservoir, such that when the at least one second valve pin is pushed, the fluid flow through the at least one second channel is occluded, and when the at least one second valve pin is released, the fluid flow through the at least one second channel is allowed; and an actuator having an actuator head for pushing or releasing the at least one first valve pin and the at least one second valve pin, such that when the gravity perfused bioreactor system is tilted in one direction, the actuator head pushes the at least one first valve pin and releases the at least one second valve pin so as to occlude fluid flow through the at least one first channel and allow fluid flow through the at least one second channel, and when the gravity perfused bioreactor system is tilted in an opposite direction, the actuator head releases the at least one first valve pin and pushes the at least one second valve pin so as to allow fluid flow through the at least one first channel and occlude fluid flow through the at least one second channel.
In one embodiment, the actuator is a rocker
In one embodiment, a rigid support is coupled to the rocker at an end opposite the actuator head for alignment with the center of the rocker.
In one embodiment, the rocker is an inverted pendulum that is bistable and movable from one side to the other as the system is tilted back and forth.
In yet another aspect, the invention relates to a gravity perfusion bioreactor system, comprising: an enclosure for cell cultures, comprising a slidable drawer/carrier; a plurality of fully enclosed, tilted reservoirs disposed in the enclosure for long-duration gravity perfusion of multiple independent bioreactors; and a modular array of gravity fed multiple independent bioreactors supported by a slidable drawer/carrier, using gravity and vertical separation to maintain consistent flow to the multiple independent bioreactors without requiring electrical power.
In one embodiment, each module includes two NVUs resting in a containment tray.
In one embodiment, fluid flows from supply containers through tubing to inputs of the multiple independent bioreactors and from outputs of the multiple independent bioreactors through tubing to waste containers.
In one embodiment, the enclosure has an HEPA filter window for atmosphere exchange, or is sealed to prevent contamination.
In one embodiment, the reservoirs comprise bag perfusion and collection reservoirs, wherein the system utilizes gravity to provide near-constant perfusion rates over for very long experiments using gas-permeable bags.
In one embodiment, the reservoirs are angled so as to maximize useful fluid volume while minimizing pressure differential as the fluid drains through the experiment.
In one embodiment, the supply and collection vials are arrayed beneath the NVU pucks.
In one embodiment, the supply and collection vials are vented and tilted.
In one embodiment, the multiple independent bioreactors are a high-density array of bioreactors with a motorized multi-channel pump running off a sealed gel lead-acid storage battery.
These and other aspects of the invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
The invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting and/or capital letters has no influence on the scope and meaning of a term; the scope and meaning of a term are the same, in the same context, whether or not it is highlighted and/or in capital letters. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
It will be understood that when an element is referred to as being “on” another element, it can be directly on the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being “directly on” another element, there are no intervening elements present. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
It will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below can be termed a second element, component, region, layer or section without departing from the teachings of the invention.
It will be understood that when an element is referred to as being “on,” “attached” to, “connected” to, “coupled” with, “contacting,” etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on,” “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” to another feature may have portions that overlap or underlie the adjacent feature.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” or “has” and/or “having” when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
Furthermore, relative terms, such as “lower” or “bottom” and “upper” or “top,” may be used herein to describe one element's relationship to another element as illustrated in the figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation shown in the figures. For example, if the device in one of the figures is turned over, elements described as being on the “lower” side of other elements would then be oriented on the “upper” sides of the other elements. The exemplary term “lower” can, therefore, encompass both an orientation of lower and upper, depending on the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as “below” or “beneath” other elements would then be oriented “above” the other elements. The exemplary terms “below” or “beneath” can, therefore, encompass both an orientation of above and below.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, “around,” “about,” “substantially” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the terms “around,” “about,” “substantially” or “approximately” can be inferred if not expressly stated.
As used herein, the terms “comprise” or “comprising,” “include” or “including,” “carry” or “carrying,” “has/have” or “having,” “contain” or “containing,” “involve” or “involving” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
As used herein, the phrase “at least one of A, B, and C” should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. The description below is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. The broad teachings of the invention can be implemented in a variety of forms. Therefore, while this invention includes particular examples, the true scope of the invention should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. For purposes of clarity, the same reference numbers will be used in the drawings to identify similar elements. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the invention.
Often gravity-induced flow through a microfluidic network or a bioreactor is a practical and very inexpensive alternative to using complicated and bulky pumps. For applications that do not require fast flows or repeated adjustments of the flow rate, or where the use of external pumping devices is cost- or space-prohibitive, gravity-induced flow offers a simple solution. The chip can contain the supply and collection reservoirs and be tilted to provide the height difference between the two; without tilting, open, vertical pipette tips can be inserted into the fluidic channels, or, also without tilting, cylindrical glass reservoirs can be bonded to the chip to deliver fluid and store waste, with the flow rate depending upon the height difference between the supply and waste reservoirs.
The primary advantage of gravity perfusion of microfluidic bioreactors is that it does not require electrical power or mechanical pumps or external tubing.
Neither system addresses the need for a compact, readily contained bioreactor perfusion system suitable for operation in a BSL-3 or BSL 4 environment as is required for the study of infectious agents and their interactions with cells cultured in microfluidic microbioreactors.
In the current COVID-19 pandemic arising from infections by the SARS-CoV-2 virus, in vitro models of the blood-brain, airway-lung, and gut barriers are of particular interest, but these measurements must be made in a Biosafety Level 3 (BSL-3) containment environment, which places severe technical and economic and space constraints on any instrumentation that is needed for the studies. For each of these physiological barriers in vivo, the structural, and biological integrity of the barrier membrane is critical to prevent infectious pathogens from crossing from an external environment to the internal, cellular environment where the infectious pathogens can multiply and spread. In a typical in vitro microfluidic model of one of these barriers, a semipermeable membrane is encapsulated between two bioreactor chambers, with one cell type on one side and a different cell type on the other.
In the case of the neurovascular unit (NVU) and the blood-brain barrier (BBB) that it protects, the barrier is formed by human brain microvascular endothelial cells on one side of a thin, porous membrane, and astrocytes and pericytes on the other. It is important that the vascular media have a high linear velocity to ensure proper biological polarization of the endothelial cells to create a tight BBB. In an in vitro airway-on-a-chip or an alveolar chip model, one side of the barrier has a confluent layer of the appropriate pulmonary epithelial cells, while the other side of the barrier has a confluent layer of pulmonary microvascular cells. In these lung-chip models, once the epithelial endothelial cell cultures are established, the perfusion liquid is removed from the epithelial side to create an air-liquid interface that can be used for aerosol infection of the chip with the SARS-CoV-2 or another virus. While in vitro models of the lining of the small intestine require a liquid-liquid interface across the endothelial cells, in vitro models of the colon may, in certain circumstances, require an air-liquid interface between the luminal and stromal sides of the interface. Other barrier chips, such as ones recapitulating the blood-testis barrier or the blood-skin barrier, would follow similar design principles, and a model of the fetal-maternal interface might require three barriers and four chambers. The embodiments presented in this disclosure are not meant to be limited to a particular organ model, interface nature, or number of chambers and barriers.
The strict protocols associated with studying infectious microorganisms in a BSL-3 facility require that all fluid-containing devices have secondary confinement.
We have successfully used gravity perfusion in the past for long-term cell culture in a thick tissue bioreactor (TTB). A simple way to control the flow rate in the gravity driven system for a given pressure differential without tilting is to have control over fluidic resistance of the network. This approach was implemented in our TTB by introducing a constricting output manifold. By selecting a manifold with appropriate internal channel dimensions, one can produce a sufficient resistance to flow that, in combination with the fixed height of the supply reservoir, could be used to passively limit the flow through the bioreactor. Such an approach works well for a given fluidic configuration but lacks flexibility and does not guarantee uniform flows from the manifold ports. An adjustable valve that can change fluidic resistance of the flow path depending on experimental requirements would be a significant improvement and a more universal solution to the passive flow regulation problem.
We envision that such a valve coupled with a gravity (or pressure) induced flow bioreactor would be opened, adjusted, or closed only once or twice during the length of the experiment. As such this valve should be simple and easily integrated with microfluidics, have minimal footprint, and be compatible with passive/stand-alone operation without external power sources and capable of reversibly opening and closing the channels. Operational modes would include 1) completely open for device fluid and cell loading or flushing; 2) set to particular predetermined position (value) based on calibration curves and experimental parameters; and 3) closed to stop the flow, for example at the end of an experiment.
One of the objectives of this invention is to disclose a new class of gravity or pressure-perfused bioreactors that are designed specifically for use in a BSL-3 or BSL-4 environment, but that have a large number of other possible applications in drug discovery, toxicology, biology, and medicine, to meet the above-noted needs.
The rate of fluid flow in gravity perfused devices is typically controlled by adjusting the difference in the height of the fluid in the input and output reservoirs. If the vertical height of the device is limited, for example by the need for a secondary containment enclosure, then the rate of flow control can be adjusted only by changing the channel width or height within the device. If high flow rates are required, the reservoirs need to have a large volume to provide perfusion fluid for extended intervals, for example one day. If large changes in flow rate are to be avoided, the reservoir should have a large horizontal cross-sectional area so that the vertical change in fluid heights is small. While not typically used in gravity perfusion, there are a large number of different microfluidic valves that operate with either mechanical or pneumatic force to compress a channel to control flow under pumped or pressure-driven flows. In many of these valves, the applied force is perpendicular to the plane that contains the bond between an elastomeric body or membrane and a rigid substrate or channel structure, thereby presenting a force that could weaken or fracture a bond required to seal the microfluidic device against leaks. The rotary via valve described in this application is unique in that it applies a horizontal, radial force against a vertical via, so that the primary compressive forces are parallel to and removed from the bonding plane, thereby producing a valve that is less likely to rupture from repeated use or overpressurization. The advantage of this valve, when coupled with integrated supply and waste reservoirs, is that the flows can be adjusted or transiently turned off as needed for a particular MPS protocol without the need to construct a microfluidic chip that is tailored for a particular flow rate.
In one aspect, the invention relates to a vertical-via rotary valve (VVRV) comprising: a valve body having a housing; one or more fluidic channels, each fluidic channel having a vertical channel portion defined in the valve body and being adjacent to the housing, wherein a fluid flow through the vertical channel portion is controllable by deforming a sidewall of the vertical channel portion; and an actuator received in the housing and rotatably engaged with the one or more fluidic channels to operably control the fluid flow through the vertical channel portion of each fluid channel.
In certain embodiments, the valve body is formed of an elastomeric material including polydimethylsiloxane (PDMS).
In certain embodiments, the vertical channel portion of each fluidic channel is a vertical via.
In certain embodiments, the actuator features a vertical trough aligned with the vertical via, such that when the actuator un-pinches the vertical via and the fluid flow through the vertical via is allowed, and when the actuator rotates from the un-pinched position, the vertical via is pinched closed and the fluid flow is restricted.
In certain embodiments, the vertical channel portions of the one or more fluidic channels in the valve body are formed in a single layer or multiple layers in the valve body.
In certain embodiments, each channel may include horizontal portions extending from and/or fluidic coupled to the vertical channel. The vertical channel and horizontal portions of the one or more fluidic channels in the valve body are formed in a single layer or multiple layers in the valve body.
In certain embodiments, the vertical channel parts are only in the central portion. The channels that lead into or out of the vertical via could be in the other layers. That said, one might make a vertical via valve that in fact requires two body layers.
In certain embodiments, both the vertical and horizontal channels are in the central portion.
In certain embodiments, the vertical channel portion of each fluidic channel is in fluid communication with a first port and a second port.
In certain embodiments, the actuator comprises one or more rolling members, and a driving member for driving the one or more rolling members to rotate independently and/or coordinately, such that when actuated, the one or more rolling members selectively address the vertical channel portions of the one or more fluidic channels so as to control rates of the fluid flow through the vertical channel portions of the one or more fluidic channels independently and/or coordinately.
In certain embodiments, the one or more rolling members comprise one or more actuating discs, wherein each actuating disc is formed with a tapered or raised protrusion such that rotation of said actuating disc results in the vertical channel portion of a corresponding fluidic channel in a fully closed state, a partially closed state, or a fully open state, depending upon a position of said tapered or raised protrusion relative to the vertical channel portion.
In certain embodiments, the one or more actuating discs are concentrically stacked to one another.
In certain embodiments, the driving member comprises one or more nesting wrenches.
In certain embodiments, the one or more rolling members comprise a plurality of rollers or a plurality of planetary gears rotatably engaged with the driving member.
In certain embodiments, the driving member comprises a top plate, a bottom plate and a central hub connected to central portions of the top plate and the bottom plate, and wherein the plurality of rollers is rotatably affixed to edge portions of the top plate and the bottom plate, such that rotation of the central hub rotates causes each roller to rotate around its own axis but not translate relative to the top plate and the bottom plate.
In certain embodiments, the driving member comprises a top plate, a bottom plate, a driving shaft connected to central portions of the top plate and the bottom plate, and a sun gear affixed to the driving shaft and situated between the top plate and the bottom plate, wherein each planetary gear is meshed with the sun gear and rotatably affixed between the top plate and the bottom plate, such that the rotation of the drive shaft is transferred to the sun gear, which in turn causes each planetary gear to rotate around its own axis but not translate relative to the top plate and the bottom plate.
In certain embodiments, the actuator comprises: one or more actuating pins accommodated in a pin housing and corresponding to the vertical channel portions of the one or more fluidic channels, wherein each actuating pin is movable from a first position in the pin housing to a second position out of the pin housing when pushed, otherwise, said actuating pin returns from the second position to the first position, wherein when said actuating pin is in the first position, it un-pinches a corresponding vertical channel portion so that the fluid flow therethrough is not restricted, and when said actuating pin is in the second position, it pinches the corresponding vertical channel portion so that the fluid flow therethrough is restricted; a pin actuator cam having at least one lobe; and a driving shaft engaged with the pin actuator cam to operably rotate the pin actuator cam, such that when the lobe is aligned with an actuating pin, the lobe pushes said actuating pin to move from the first position to the second positon, thereby restricting the fluid flow through said corresponding vertical channel portion, when the lobe is misaligned with said actuating pin, said actuating pin returns from the second position to the first position, thereby, unrestricting the fluid flow through said corresponding vertical channel portion.
In certain embodiments, each actuating pin is accommodated in the pin housing with an elastic member including a spring.
In certain embodiments, the return force on the actuator pin is provided by the elastomeric material of the valve body, including PDMS.
In certain embodiments, the VVRV is a mixing valve.
In certain embodiments, the valve body is disposable, while the actuator is never in contact with fluid and is reusable.
In another aspect, the invention relates to a gravity perfused bioreactor, comprising: a plurality of chambers vertically stacked to one another on a substrate; and a plurality of reservoirs disposed over the plurality of chambers, wherein each chamber has an input port and an output port fluidically coupled to two corresponding reservoirs of the plurality of reservoirs, respectively; and wherein the plurality of reservoirs and the plurality of chambers are configured such that fluid flows between the plurality of reservoirs and the plurality of chambers are gravity-fed.
In certain embodiments, the gravity perfused bioreactor is devoid of a valve.
In certain embodiments, the gravity perfused bioreactor further comprises a plurality of permeable membranes, wherein each permeable membrane is disposed between two vertically adjacent chambers.
In certain embodiments, the plurality of chambers comprises an endothelial chamber and an epithelial chamber.
In yet another aspect of the invention, the gravity perfused bioreactor comprises: a ribbon fluidics comprising supply channels, collection channels, and a fluid manifold fluidically coupled between the supply channels the collection channels; a bioreactor disk attached to the ribbon fluidics, comprising a bioreactor fluidically coupled to the fluid manifold of the ribbon fluidics; a plurality of supply reservoirs fluidically coupled to the supply channels of the ribbon fluidics; and a plurality of collection reservoirs fluidically coupled to the collection channels of the ribbon fluidics, wherein the plurality of supply and collection reservoirs and the bioreactor are configured such that fluid flows between the plurality of supply and collection reservoirs and the bioreactor are gravity-fed.
In certain embodiments, the gravity perfused bioreactor is devoid of a valve.
In certain embodiments, each reservoir comprises an intubation recess.
In certain embodiments, the ribbon fluidics comprises an inverted, twisted ribbon fluidics.
In certain embodiments, the ribbon fluidics is adapted for invertible loading.
In certain embodiments, the supply reservoirs, the bioreactor, and the collection reservoirs are terraced for passive pumping.
In certain embodiments, the gravity perfused bioreactor further comprises an openable puck housing and an openable puck compression ring coupled to the bioreactor.
In certain embodiments, the bioreactor is mounted on a microscope slide.
In certain embodiments, the supply reservoirs are situated at a higher elevation than the collection reservoirs.
In certain embodiments, the bioreactor comprises a plurality of bioreactor layers vertically stacked to one another, which in turn is detachably attached to the fluid manifold.
In certain embodiments, the gravity perfused bioreactor further comprises a plurality of VVRVs. Each VVRV is fluidically coupled between one of the supply reservoirs and the collection reservoirs and the bioreactor.
In certain embodiments, the gravity perfused bioreactor further comprises at least one pump for inducing the fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers.
In certain embodiments, the gravity perfused bioreactor further comprises external pressure or suction sources including mechanical pumps or external gravity-feed/collection reservoirs.
In certain embodiments, the flow rates and pressures are adjustable on-the-fly by changing the relative pressure differential between the supply and collection reservoirs by adjusting the relative elevation of supply fluid of the supply reservoirs to collection fluid of the collection reservoirs.
In certain embodiments, the flow rates and pressures are adjustable by changing perfusate volume in the reservoirs or elevating the supply reservoirs relative to the collection reservoirs.
In a further aspect of the invention, the gravity perfused bioreactor comprises: a plurality of bioreactor chambers stacked to one another, wherein each chamber has an input port and an output port; a plurality of reservoirs disposed over the plurality of bioreactor chambers; and a plurality of VVRVs. Each VVRV is disclosed above and fluidically coupled between a corresponding reservoirs of the plurality of reservoirs and one of the input port and the output port of a corresponding chamber of the plurality of bioreactor chambers.
In certain embodiments, the gravity perfused bioreactor further comprises a plurality of permeable membranes, wherein each permeable membrane is disposed between two adjacent bioreactor chambers.
In certain embodiments, fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers are gravity-fed, and/or induced by mechanical pumping.
In certain embodiments, the gravity perfused bioreactor further comprises at least one pump for inducing the fluid flows between the plurality of reservoirs and the plurality of bioreactor chambers.
In certain embodiments, the gravity perfused bioreactor further comprises external pressure or suction sources including mechanical pumps or external gravity-feed/collection reservoirs.
In one aspect of the invention, the gravity perfused bioreactor system comprises: at least one bioreactor chamber; and a plurality of reservoirs, wherein the plurality of reservoirs comprises an autofilling supply reservoir, a bioreactor supply reservoir, and a bioreactor collection reservoir; wherein the at least one bioreactor chamber is fluidically coupled between the bioreactor supply reservoir and the bioreactor collection reservoir; and wherein the autofilling supply reservoir is fluidically coupled to the bioreactor supply reservoir by a reservoir delivery tube and a level-sensing air vent and configured with adjustable level control for a constant pressure head, such that media in the autofilling supply reservoir is released only when a fluid level in the bioreactor supply reservoir drops below the lower end of the level-sensing air vent, at which point air enters the autofilling supply reservoir, increases a volume of air trapped in the autofilling supply reservoir, and allows fluid to move from the autofilling supply reservoir to the bioreactor supply reservoir through the reservoir delivery tube.
In certain embodiments, the media from the bioreactor supply reservoir then flows through the at least one bioreactor chamber into the bioreactor collection reservoir, thereby increasing the level of media in the bioreactor collection reservoir.
In certain embodiments, the gravity perfused bioreactor system further comprises a vertical adjustment means coupled to the autofilling supply reservoir for setting the fluid height in the bioreactor supply reservoir.
In certain embodiments, the autofilling supply reservoir is situated over the bioreactor supply reservoir, or located remote from the bioreactor supply reservoir.
In certain embodiments, the gravity perfused bioreactor system further comprises a wick fluidically coupled to the bioreactor collection reservoir for adjusting the fluid level in the bioreactor collection reservoir.
In certain embodiments, the wick comprises a collection bag, a collection tube having one end placed in the bioreactor collection reservoir and the other end placed in the collection bag, and a collection wick received in the collection tube.
In certain embodiments, the gravity perfused bioreactor system further comprises a siphon tube inserted in a side punched hole of the bioreactor collection reservoir for adjusting the fluid level in the bioreactor collection reservoir.
In certain embodiments, the autofilling supply reservoir is a large-volume airtight reservoir that maintains a constant supply reservoir height therein, and the wick or tube collection system ensures that the collection fluid in the bioreactor collection reservoir has a near-constant height so that media flows from the airtight autofilling reservoir, through the bioreactor supply reservoir, and perfuses the bioreactor at a constant flow rate.
In another aspect of the invention, the gravity perfused bioreactor system comprises: a first reservoir and a second reservoir; at least one bioreactor chamber having at least one first channel fluidically coupled to the first reservoir, and at least one second channel fluidically coupled to the second reservoir; and a valve comprising: at least one first valve pin situated on the at least one first channel between the first reservoirs and the at least one bioreactor chamber, such that when the at least one first valve pin is pushed, fluid flow through the at least one first channel is occluded, and when the at least one first valve pin is released, the fluid flow through the at least one first channel is allowed; at least one second valve pin situated on the at least one second channel between the at least one bioreactor chamber and the second reservoir, such that when the at least one second valve pin is pushed, the fluid flow through the at least one second channel is occluded, and when the at least one second valve pin is released, the fluid flow through the at least one second channel is allowed; and an actuator having an actuator head for pushing or releasing the at least one first valve pin and the at least one second valve pin.
As such, when the gravity perfused bioreactor system is tilted in one direction, the actuator head pushes the at least one first valve pin and releases the at least one second valve pin so as to occlude fluid flow through the at least one first channel and allow fluid flow through the at least one second channel, and when the gravity perfused bioreactor system is tilted in an opposite direction, the actuator head releases the at least one first valve pin and pushes the at least one second valve pin so as to allow fluid flow through the at least one first channel and occlude fluid flow through the at least one second channel.
In certain embodiments, the actuator is a rocker
In certain embodiments, a rigid support is coupled to the rocker at an end opposite the actuator head for alignment with the center of the rocker.
In certain embodiments, the rocker is an inverted pendulum that is bistable and movable from one side to the other as the system is tilted back and forth.
In yet another aspect of the invention, the gravity perfused bioreactor system comprises: an enclosure for cell cultures, comprising a slidable drawer/carrier; a plurality of fully enclosed, tilted reservoirs disposed in the enclosure for long-duration gravity perfusion of multiple independent bioreactors; and a modular array of gravity fed multiple independent bioreactors supported by a slidable drawer/carrier, using gravity and vertical separation to maintain consistent flow to the multiple independent bioreactors without requiring electrical power.
In certain embodiments, each module includes two NVUs resting in a containment tray.
In certain embodiments, fluid flows from supply containers through tubing to inputs of the multiple independent bioreactors and from outputs of the multiple independent bioreactors through tubing to waste containers.
In certain embodiments, the enclosure has an HEPA filter window for atmosphere exchange, or is sealed to prevent contamination.
In certain embodiments, the reservoirs comprise bag perfusion and collection reservoirs, wherein the system utilizes gravity to provide near-constant perfusion rates over for very long experiments using gas-permeable bags.
In certain embodiments, the reservoirs are angled so as to maximize useful fluid volume while minimizing pressure differential as the fluid drains through the experiment.
In certain embodiments, the supply and collection vials are arrayed beneath the NVU pucks.
In certain embodiments, the supply and collection vials are vented and tilted.
In certain embodiments, the multiple independent bioreactors are a high-density array of bioreactors with a motorized multi-channel pump running off a sealed gel lead-acid storage battery.
In certain embodiments, a gravity perfused bioreactor can be seeded initially upright and/or inverted by a tubing connection to a larger gravity reservoir, a syringe pump, or an external peristaltic pump and then, after the tubing is removed from the bottom of the reservoirs, the device can be inverted and the now-upright reservoirs can be filled to support gravity perfusion.
In certain embodiments, a gravity perfused airway bioreactor has reservoirs on opposite sides so that the cells on the airway side can be perfused during and after seeding, and the device then inverted to operate the airway side as an air-liquid interface while the opposite, endothelial side is gravity perfused and seeded.
In certain embodiments, a vertical-via rotary valve (VVRV) allows regulation of the rate of flow from an integral supply reservoir through an MPS microbioreactor into a collection reservoir.
In certain embodiments, the valve body is designed to be disposable, single use, while the actuator, which is never in contact with fluid, can be reused.
In certain embodiments, a VVRV provides independent control of multiple fluid flows.
In certain embodiments, a VVRV provides coupled control of two or more fluid flows to comprise a mixing valve.
In certain embodiments, a VVRV utilizes rollers within the VVRV actuator to minimize friction.
In certain embodiments, a VVRV has an eccentric cam actuator.
In certain embodiments, a VVRV uses actuator pins.
In certain embodiments, a VVRV actuator is geared to provide both additional torque and finer control of flow rate.
In certain embodiments, a gravity perfused bioreactor with one, two, or more chambers, separated by semipermeable membranes, is connected to integrated perfusion reservoirs by a flexible ribbon fluidic that allows the orientation of the bioreactor to be inverted without inverting the reservoirs, and allows the relative height of the reservoirs to be adjusted to control flow rate.
In certain embodiments, a multi-chamber bioreactor can be permanently bonded or reversibly clamped to a flexible-fluidic gravity perfused manifold, so that the design and fabrication of the bioreactor and the fluid manifold are decoupled.
In certain embodiments, in a gravity perfused bioreactor, the level of fluid in the supply reservoir is maintained by a second reservoir from which fluid flows based upon the height of fluid in the supply reservoir.
In certain embodiments, in a gravity perfused bioreactor, the level of fluid in the collection reservoir is determined by the height of a wick in the collection reservoir, with the wick delivering the collected fluid into a lower secondary collection reservoir or bag.
In certain embodiments, a gravity perfused tilting bioreactor for which the direction of flow between two reservoirs is controlled by a valve whose operation is controlled by the tilting operation to provide unidirectional flow through the bioreactor.
In certain embodiments, a system of gravity perfused bioreactors can be readily enclosed in a secondary containment enclosure.
In certain embodiments, a system of gravity perfused bioreactors uses tilted supply and collection reservoirs or bags to minimize height differences during prolonged perfusion while enclosed in a secondary containment enclosure.
In certain embodiments, a system of battery-powered pumped perfused bioreactors can be readily enclosed in a secondary containment enclosure and operated for several days without intervention.
In certain embodiments, a perfusion system accommodates both passive (gravity-fed) and active (mechanical or other active pumping) perfusion.
In certain embodiments, a perfusion system accommodates integrated (on-board, self-contained) gravity-promoted flow and also accommodates external pressure or suction sources (such as mechanical pumps or external gravity-feed/collection reservoirs).
In certain embodiments, a gravity-fed perfusion system allows flow rate and pressure to be adjusted on-the-fly by changing the relative pressure differential between supply and collection reservoirs by adjusting the relative elevation of supply fluid to collection fluid. This may be accomplished by changing perfusate volume in the reservoirs or elevating the supply reservoir relative to the collection reservoir.
These and other aspects of the invention are further described below. Without intent to limit the scope of the invention, exemplary valves, bioreactor systems, methods and their related results according to the embodiments of the invention in conjunction with the accompanying drawings in
Noting the parallel between the design requirements of NVU, gut, and airway barriers, for the remainder of this description we will for simplicity and clarity focus our embodiments on an airway model, but the design features and principles presented herein will apply to other organ chips as well.
Following the design concept shown in
A requirement for perfusion of the airway chip and other universal gravity-fed bioreactors is the ability to adjust the flow rate through each layer of a two-chamber bioreactor independently. One of the challenges with gravity-fed bioreactors is that there are only two ways to adjust the flow rate through the bioreactor: by controlling the height difference between the supply and collection reservoirs, and by adjusting the channel width in the device to reduce or increase the hydraulic resistance of the fluid network connecting the two reservoirs. The former is straightforward but tedious and may limit the dynamic range over which the reservoir heights can operate. The latter involves the fabrication of a new chip and allows a chip to have a single flow rate, rather than different flow rates at different times, e.g., high flow rates for cell loading and low flow rates for perfusion. To provide the capability to control fluidic resistance without fabricating a new chip, we present a very simple mechanical valve that can be used to regulate the hydraulic resistance without physically modifying microfluidic chip or the bioreactor therein.
Note that the channels on both sides of 611 can be embossed or molded into either the upper and lower surfaces of the middle slab 607 or in the lower surface of the upper membrane 606, or the upper surface of the lower membrane 605, or combinations thereof. The three layers, 605, 607, and 606, should be bonded to each other, either by thermal or chemical means, to make the channels leak-tight. Note that in this device, the fluid-carrying channels do not make direct contact with the actuator 641, whereas in prior art shown in
In the situation shown in
The operating principle of the VVRV with a narrow actuating trough 642 is depicted in
The key feature of the trough is that it provides an actuator with different radial distances from the axis of rotation: the deepest part of the trough is designed so that a vertical via adjacent to that spot will be open. Outside the trough, the radius of the actuator is such that when a vertical via is adjacent to a region with the larger radius, the via will be compressed and sealed. As shown in
The operating principle of the VVRV with a tapered actuating protrusion is depicted in
The actuator 641 is enclosed by the body of a vertical-via rotating valve comprising layers 605, 606, and 607. The actuator cylindrical bearing surface 644 maintains the alignment of the actuator 641 in the hole 652 in the valve body to allow precise, low-friction rotation of the valve actuator. The compression region 654 is deflected by the actuator protrusion 627 to partially close the via 628. A drive shaft 661 for the actuator for the vertical-via rotating valve controls the angle of the actuator and its protrusions relative to the via.
Another advantage of the vertical-via rotary valve is that the configuration of the actuator protrusions can be adjusted without having to alter the fluidic chip and its microfluidic channels and vias. For example, as shown in
Referring to
This third embodiment of the VVRV was designed with precision adjustment of flow rate in mind. We call this version the vertical-via rotary metering valve. The radial protrusion of the cam gradually increases from zero, i.e., open to closed, as the actuator is rotated up to 180°, allowing the user to fine-tune the magnitude of occlusion seen by the via.
It should be clear that the compressive forces that close the via are radial, do not apply a separating force directly to a bonded layer, and provide continuous metering whose angular dependence can be specified by the design of the protruding cam.
As shown in
The merit of this design is that the recesses shown in
In a comparison of the operation of different actuator designs that we have presented,
In
While the valve body shown in
Another important aspect of this design is that it can scaled to different sizes; the actuators in current designs are 10 mm in diameter; larger and smaller designs could be readily implemented.
We have identified a further application of the vertical-via rotary metering valve concept, which is to incorporate multiple vias whose flow rates are controlled by a single metering actuator.
One embodiment of this approach is shown in
The embodiment shown in
To the inventors' best knowledge, no other microfluidic valve that with a single actuator can provide such mixing.
Independent control of fluid flow through multiple vertical vias on a single chip may be accomplished using an embodiment of independent coaxial actuator assembly 1301 shown in
Alternatively, two coaxial actuator disks can have staggered surfaces such that the body of one is above the other, but the actuator lobe on each is coplanar, so that the two lobes of a single actuating plane can be adjusted independently. A locking screw can hold them at a fixed relative position. The embodiments shown herein are not meant to imply in any way a limitation in the arrangement and control of one or more actuator protrusions or troughs.
Other embodiments allow for a variety of different actuation configurations.
The concept of rotary actuators can be expanded by creating a stack of coaxial actuators that can each actuate one or more of a multiplicity of vias surrounding a common hole in the valve body, with each via being controlled independently.
As described elsewhere in this invention, actuator protrusions 1553 may be positioned relative to valved vias 1523 such that wall 1524 is partially deformed, in which case flow through via 1528 is throttled as in
While this embodiment can have actuator recesses around the central cavity to keep the actuating disks in place, assembly of the valve would be simplified without them. It is important to realize that each via needs to be compressed by an actuator protrusion, and hence can either be configured as shown in
As described elsewhere in this invention, actuator protrusions may be positioned relative to valved via 1628 such that the wall between actuator 1603 and via 1628 is partially deformed, in which case flow through via 1628 is throttled; completely occluded, in which case flow through via 1629 is completely prevented; or not deformed, in which case the via remains unconstricted (not shown).
It is important to recognize that other than TURN or Twist valves, the vast majority of microfluidic valves are either open or closed, with little ability to throttle the output. TURN and Twist valves use a threaded screw to apply a downward force to close a horizontal channel. Overtightening the screw can debond the valve. The via valve in the present invention operates by means of a radial force that does not present a risk of valve debonding upon valve activation activation.
In a more detailed rendering of a four-channel vertical-via rotary valve, the actuator recesses that were not shown in
Note that in this illustration the compression of the elastomer between the fully closed via and the actuator is not shown in detail. In one exemplary embodiment, the actuator is similar to that shown in
One of the limitations of the actuators shown so far is that the actuator protrusion has to slide across the PDMS surface in the vicinity of the controlled via such that the PDMS is compressed to close the valve. There will be friction associated with this sliding, which in turn requires application of a torque to overcome the frictional force. An alternative to this approach is to use the roller actuators shown in
This embodiment of the VVRV uses planetary actuator 1800 as shown in
While the functional principle is the same as that previously presented, roller actuator 1800 was designed to minimize friction, as it only contacts the valve body in the immediate region of actuation 1850 and the extremities of those rollers opposite the via.
The torque required to rotate the roller actuator in
In summary,
Referring to
In recognition of the need to be able to adjust the perfusion rate of a generic gravity perfused bioreactor,
As shown in
Given the need to seed cells on both sides of a barrier membrane while creating an airway chip, we have devised a bioreactor that has reservoirs on each side so that one side can be gravity perfused prior to cell attachment, at which point it is inverted and the other side is gravity perfused.
The present system would be useful in an application wherein, for example, cells are to be seeded on a surface (e.g., a membrane separating the two bioreactor chambers) that will become a ceiling within the interior of the bioreactor chamber when the system is inverted.
Clearly, the present system can be used in gravity-feed mode only for the circuit connected to the upward-facing reservoir pair. However, as shown in
In other embodiments of the spring-return-based valve, the surface of actuator cam 2393 that contacts pins 2391 may take various shapes, as illustrated by
The spring force that returns pins 2391 from their extended positions to their relaxed state may be provided by the elastomeric material from which the valve body 3211 is constructed, as in
Step 1: Load endothelial side while inverted and maintain cells with pumped perfusion for one day. Both sides are perfused with pumps since this is not performed in BSL-3.
Step 2: Load epithelial side while right side up, maintaining both sides with pumped perfusion for one more day. Both sides are perfused with pumps as this is not performed in BSL-3.
Step 3: Remove tubing from inlet of epithelial side and pump to remove media from the entire airway chamber to establish a mature air-liquid interface.
Step 4: Invert the device with reservoirs up and seed the endothelial side. Perfuse with pumping until endothelium is mature.
Step 5: Remove tubing from outlet side of endothelial chamber to allow some perfusate to fill the tubing port.
Step 6: Remove the tubing from the inlet port and fill with required media for duration of experiment. Remove tubing from epithelial outlet.
It also may be possible to avoid the inverted tubing-perfusion steps as long as the cells in the lower chamber are immersed in trapped fluid without perfusion for only the several hour interval required to attach the cells on the other (then up) side of the barrier.
An alternative means to the inversion-for-cell-seeding step discussed above is to use ribbon fluidics to invert the bioreactor while leaving the reservoirs stationary.
Bioreactor layers 2618, 2619 may be bonded together or otherwise clamped or affixed to each other, and in turn this assembly may be bonded to manifold 2631, or the whole stack may be clamped together and to manifold 2631, making the bioreactor assembly detachable from the perfusion system.
An alternative intermediate between a fixed-design gravity bioreactor as shown in
A limitation of the gravity perfusion systems discussed so far is that the level in the supply reservoir drops with time while that in the collection reservoir rises, leading to a steady reduction in the pressure differential between the two sides and a corresponding decrease in flow rate.
The media 2808 in the airtight autofilling supply reservoir 2855 is released only when the fluid level in the bioreactor supply reservoir 2880 drops below the lower end of the level-sensing air vent 2856, at which point air enters autofilling reservoir 2855, increases the volume of air 2897 trapped in autofilling reservoir 2855, and allows fluid to move from the autofilling reservoir 2855 to the bioreactor supply reservoir 2880 through the reservoir delivery tube 2857. Media from the bioreactor supply reservoir 2880 then flows through bioreactor chamber 2870 into bioreactor collection reservoir 2885, increasing the level of media 2898 in that reservoir. An unspecified vertical adjustment means 2858 is used to set the fluid height in the bioreactor supply reservoir 2880. Note that the dual-tube feeder design (2857 and 2856) allows for smaller tubing and a variable length that can allow reservoirs to be remotely located.
On the collection side, the height of fluid in the collection reservoir would normally increase with time and thereby reduce the flow rate through the bioreactor.
Alternatively, as shown in
Together, the large-volume airtight reservoir 2895 maintains a constant supply reservoir height 2895, and the wick or tube collection system ensures that the collection fluid 2896 has a near-constant height so that media 2896 flows from the airtight autofilling reservoir, through the bioreactor supply reservoir, and perfuses the bioreactor at a constant flow rate.
Long-term gravity perfusion of bioreactors is often performed using a tilt table that allows the media to flow back and forth between two reservoirs, in which case the reservoir that is high is the supply reservoir that delivers media to the lower, collection reservoir. When the media in the supply reservoir is depleted, the bioreactor is tilted, so that the functions of the two reservoirs is reversed: supply becomes collection, and collection become supply. The biologically significant limitation of this approach is that the direction of perfusion in the vascular space reverses between the two cycles, and the proper polarization, for example, of the brain microvascular endothelial cells critical for proper performance of the blood-brain barrier is compromised, leading to increased barrier permeability. It is possible to build a fluidic chip that has small, on-board reservoirs and a channel system that supports unidirectional flow without valves. An alternative approach that can support more generalized reservoir and channel design is presented in
As we have stated earlier, the study of infectious micro-organisms requires not only BSL-3 facilities but also secondary containment. A commercial means for accomplishing this in a standard cell culture environment is to use systems such as the Thermo Fisher Scientific Cell Locker®, a HEPA filtered enclosure for cell culture.
Designed specifically to maximize usable space within the Thermo Fisher Scientific Cell Locker®, the three modules shown in assembly 3000 support cell experiments (each shown with two NVUs resting in Petri dishes or another such containment tray 3004) using gravity and vertical separation to maintain consistent flow to the experiment(s) without requiring electrical power. As shown, these modules provide space for up to 4 standard input syringe vials (with vented plugs) and up to 4 collection vials per module.
The tilted reservoirs provide long-duration gravity perfusion while minimizing the changes in fluid level during the course of the experiment. The HEPA filter window 3001 provides atmosphere exchange between the modules and the incubator in which they are operated. The unit is otherwise sealed to prevent contamination.
The modular array of gravity fed experiments 3002 is supported by sliding drawer/carrier 3005. In this embodiment, each module is shown carrying two experiments within a Petri dish 3004 and fed by the pressure differential caused by the vertical separation of the syringe bodies and the experiments and the experiments to the waste vials 3006 stored underneath. These containers and orientation may vary. Modules shown have optional storage for extra fluid containers both above and below experiments in Petri dish 3004.
The fluid supply reservoir 3003, here represented by a capped and vented syringe body, is angled so as to maximize useful fluid volume while minimizing pressure differential as the fluid drains through the experiment. Fluid flows from supply containers 3003 through tubing (not shown) to the inputs of experiments 3004 and from the experiment outputs through tubing (not shown) to the waste containers 3006.
As an alternative to the vial reservoirs in
While there are merits to using gravity perfusion, it presents certain limitations, most notably the inability to recirculate media without manually moving media from the collection reservoir back to the supply reservoir, as well as there being no means to performed timed drug, toxin, or microbe delivery without opening the locker.
Using the same HEPA filtered storage drawer 3000, this embodiment is focused on maximizing the number of experiments per unit volume and carries a multi-channel pump and battery capable of running the illustrated twelve-experiment set 3101 (with 12 independent fluid channels) continuously. Supply and collection vials 3102 are arrayed beneath the NVU pucks 3101 as shown. Additional images more clearly show the pump 3104 motor and battery unit 3103. Sealed battery chemistry prevents leaks and off-gassing into the cell culture environment. Vials 3102 are vented and tilted such that a long needle is ideally placed to remove as much fluid from the container as possible. Wiring, tubing, and optional digital interface are not shown.
In conclusion, this invention provides detailed descriptions of the four major, integrated subsytems needed for BSL-3 studies of infectious organisms: bioreactors, valves, perfusion reservoirs, and secondary containment.
The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the invention pertains without departing from its spirit and scope. Accordingly, the scope of the invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
Some references, which may include patents, patent applications, and various publications, are cited and discussed in the description of the invention. The citation and/or discussion of such references is provided merely to clarify the description of the invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.
This application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 63/017,744, filed Apr. 30, 2020, which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. UH3TR002097 and 3UH3TR002097-04S1 awarded by the NIH National Center for Advancing Translational Sciences (NCATS), National Institute of Neurological Disorders and Stroke (NINDS), and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD); Grant No. U01TR002383 and (through Vanderbilt University Medical Center) UL1TR002243 awarded by NCATS; Grant No. U01CA202229 awarded by the National Cancer Institute (NCI), and Grant No. HHSN271201 700044C awarded by the National Center for Advancing Translational Sciences (NCATS) through CFD Research Corporation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/030219 | 4/30/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63017744 | Apr 2020 | US |
