Patent Application
20200308524
High aspect ratio vessels (HARV), also referred to as slow-turning lateral vessels (STLV), rotating wall vessels (RWV) and clinostat bioreactors are specific types of a rotary bioreactor that provide conditions of “simulated microgravity” (SMG) which causes cells to form aggregates, express genes differently, and induce a host of other scientifically relevant behaviors. SMG has a broad array of applications from space biology studies, to developmental biology, to tissue engineering. To generate SMG conditions, a bioreactor is filled completely with fluid and rotated such that the motion of the fluid with the cells averages out the effects of gravity. This effect is only accomplished if the chamber is entirely filled with fluid thereby effectively creating solid body rotation. Any air bubbles present can induce a “slosh dynamic” effect that introduces turbulence and harms ideal fluid dynamics. All HARVs and STLVs possess a gas permeable membrane to allow cellular respiration and to normalize pH with incubator CO2 levels. This membrane can allow for the passage of water vapor if the humidity level is not extremely high. Loss of liquid will rapidly create bubbles, sometimes within in a matter of hours. This demands constant monitoring and checking of the bioreactor and the incubator, and the formation of bubbles can destroy experiments that may take days or weeks to prepare.
Organoids and Spheroids in Rotating Wall Vessel (RWV) Bioreactors:
2D cell culture was the gold standard of in vitro biology for nearly a century and has been used in virtually every major discovery in cell biology. However, increasing understanding of the complexity of biological systems has led to heightened awareness of the importance of model systems that more closely replicate the true 3D environment of living tissues (Antoni, Burckel, Josset, & Noel, 2015). While extensive progress has been made in engineered 3D scaffolds, organ recellularization, and bioprinting, recent studies demonstrate the unique potential of self-assembled spheroids and organoids for discoveries in disease modeling, drug discovery, and a host of other biomedical applications (Xinaris, Brizi, & Remuzzi, 2015).
Although several protocols exist for the generation of spheroids and organoids, such as spontaneous self-assembly in non-adherent cell culture plate (Fang & Eglen, 2017) or the hanging drop method (Eder & Eder, 2017), culturing cells in the RWV bioreactor with its improved nutrient exchange yields consistent formation of organoids with superior morphology and unique, organotypic gene expression (Botta, Manley, Miller, & Lelkes, 2006; Gerecht-Nir, Cohen, & Itskovitz-Eldor, 2004; Lelkes et al., 1998; Mattei, Alshawaf, D 'abaco, Nayagam, & Dottori, 2018; Radtke & Herbst-Kralovetz, 2012; Redden & Doolin, 2011; Redden, Iyer, Brodeur, & Doolin, 2014). Recently, DiStefano, et al. demonstrated that a continuous perfusion variant of the RWV produced murine retinal organoids with properly organized photoreceptor cells significantly faster and larger than static cultures (DiStefano et al., 2018). Faster generation of larger high-fidelity organoids in the RWVs may facilitate the dissemination of these organoids for high-throughput tissue analysis in drug discovery and personalized medicine.
Bubble Formation in the Rotating Wall Vessel Bioreactor and Its Impact:
Initially developed by NASA in the 1980s, RWVs were originally intended to safely shuttle cells into space. However, it was quickly discovered that the RWV was also capable of supporting the formation of complex, 3D, tissue-like structures of relevance to biomedical research on the ground (Schwarz, Goodwin, & Wolf, 1992).
Because the low-shear, microgravity effect of the RWV relies on a circular, solid-body fluid path along its interior perimeter, the RWV requires a “zero headspace” condition, literally a continuous interface between the fluid and the wall with no air gap (Hammond & Hammond, 2001; Wolf & Schwarz, 1992). When this condition is violated due to the presence of a bubble, this circular path is interrupted, as buoyancy continually forces the bubble to the highest point of the system opposite the flow of the fluid. The presence of a bubble at this locale interrupts the fluid path, adding turbulence and fluid shear. Bubbles are easily formed because each RWV bioreactor has a gas exchange system for cellular respiration and to pH-balance the cell culture media with incubator CO2. When the incubator environment has insufficiently high humidity (below 100%), negative pressure can form as water vapor diffuses away from the bioreactor, inevitably causing dissolved gases to precipitate out, forming unwanted bubbles. The need to remove bubbles from the RWV system prior to use is widely discussed in literature (Hammond, Allen, & Birdsall, 2016; Hammond & Hammond, 2001; King & Miller, 2007; Pollack, Meaney, Levine, Litt, & Johnston, 2000; Radtke & Herbst-Kralovetz, 2012; Salerno-Goncalves, Fasano, & Sztein, 2016; Varley, Markaki, & Brooks, 2017; Wuest, Richard, Kopp, Grimm, & Egli, 2015). However, what is not widely discussed is the significance of failing to do so or an analysis of the change in shear due to the presence of a bubble.
Though the importance of removing initial bubbles is widely reported, a rational and consistent way of preventing bubble formation is not. While bubbles can enter the RWV system through leaking or improper clearing, the most difficult source of bubbles to prevent is due to gas exchange. Some authors suggest a sufficiently high humidity incubator (several water pans used simultaneously) will prevent bubble formation (Hammond & Hammond, 2001). However, with the need to open the incubator for media changes or removal of samples at different time points, this becomes difficult to maintain in practice, resulting in the frequent formation of small bubbles within 24-48 hours, which in turn can act as nucleation points for large bubble growth. Over longer time points, such as that required for organoid differentiation, bubble prevention becomes virtually unsustainable. With the potential for the proliferation of RWV bioreactor technology for organoid research, there is a need for an improved system capable of neutralizing the impact of incidental bubbles.
Accordingly, there is a need in the art for an improved HARV or STLV rotary bioreactor device that has means for instantly and continuously removing bubbles from the bioreactor without disruption to fluid dynamics, all while minimizing the maintenance and disruption to the operation of the rotary bioreactor. Embodiments of the invention described herein meet these needs.
In one embodiment, a culture chamber for a rotary bioreactor includes a central chamber; an elongate channel comprising a channel entrance configured to receive bubbles from the central chamber; wherein the elongate channel surrounds a majority of the central chamber. In one embodiment, the channel entrance comprises a back wall that is common to an outer wall to the elongate channel. In one embodiment, the elongate channel terminates in a channel exit port. In one embodiment, the elongate channel is curved. In one embodiment, the curvature of the elongate channel approximates a curvature of the perimeter of the central chamber. In one embodiment, the elongate channel surrounds the entire central chamber. In one embodiment, the elongate channel extends beyond one complete turn around the central chamber. In one embodiment, the channel entrance comprises a gating mechanism. In one embodiment, the gating mechanism is one of a hydrophobic membrane, a ball valve or a one-way flow valve. In one embodiment, the culture chamber is substantially disc-shaped. In one embodiment, the culture chamber is substantially drum-shaped. In one embodiment, the channel entrance is disposed at the vertex of at least one angled ramp. In one embodiment, the channel entrance is disposed between and at the vertex of at least two angled ramps. In one embodiment, the culture chamber includes an outer sheath at least partially comprising the elongate channel. In one embodiment, a culture chamber assembly includes a plurality of disks that form the culture chamber. In one embodiment, the culture chamber assembly includes a gas-permeable membrane layer. In one embodiment, the culture chamber assembly includes a gasket layer at least partially comprising one of the central chamber and the elongate channel. In one embodiment, a bioreactor system includes the culture chamber and a rotator device configured to attach to and rotate the culture chamber. In one embodiment, a HARV or STLV simulated microgravity kit includes the culture chamber. In one embodiment, the channel entrance shares the same plane as the central chamber. In one embodiment, the channel entrance and the central chamber are configured in different planes. In one embodiment, the culture chamber includes a second elongate channel comprising a second channel entrance configured to receive bubbles from the central chamber. In one embodiment, the channel entrance, the second channel entrance and the central chamber are all configured in different planes. In one embodiment, the central chamber is configured to accept a working volume of 10 mL of media or less.
In one embodiment, a method for removing bubbles from a cell culture includes the steps of introducing media into a culture chamber, continuously rotating the culture chamber, affecting the movement of bubbles from a central chamber of the culture chamber to a peripheral channel of the culture chamber as the culture chamber rotates, and maintaining the bubbles in the peripheral channel as the culture chamber rotates. In one embodiment, the step of introducing media into a culture chamber includes introducing media of 10 mL or less.
The foregoing purposes and features, as well as other purposes and features, will become apparent with reference to the description and accompanying figures below, which are included to provide an understanding of the invention and constitute a part of the specification, in which like numerals represent like elements, and in which:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods and materials are described.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “simulated microgravity” refers to the creation of a solid rotational body of fluid in which the gravitational vector is averaged to near zero for particles suspended in said fluid.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The present invention relates to novel vessels for rotary cell culture bioreactors. In some embodiments, the present invention provides novel high aspect ratio vessels (HARV) for rotary cell culture bioreactors. A HARV is a type of rotary bioreactor which can expose cells to simulated microgravity conditions. In some embodiments, the present invention provides novel slow turning lateral vessels for rotary (STLV) cell culture bioreactors. An STLV operates much like a HARV but has been extended along its axis of rotation.
Embodiments of the invention described herein actively and continuously remove gas bubbles from the system before they are able to affect bioreactor performance. A structure such as a disc housing the main volume includes a thin channel that runs around the majority of the perimeter of the disk. The channel captures bubbles that move away from the main volume during rotation, allowing the bubbles to maintain a position significantly far away from the main volume so that they do not interact with the main body of the fluid. As a result, bubbles are instantly and continually removed from the main body of fluid, there is no disruption to fluid dynamics, the system requires less observation and upkeep for experiments, experiments are less likely to fail, and incubators can run at lower humidity with fewer water trays and are less likely to experience contamination.
With reference now to
In one embodiment, the elongate channel is a curved channel. In one embodiment, the curvature of the channel is the same or close to the perimeter of the central chamber 21 and/or the perimeter of the HARV disk. In one embodiment, the elongate channel 24 runs around less than or about half the perimeter of the central chamber 21. In one embodiment, the elongate channel 24 runs around the majority of the perimeter of the central chamber 21. In one embodiment, the elongate channel 24 runs around the entire perimeter of the central chamber 21. In certain embodiments, the elongate channel 24 can continue to run past one complete turn around the perimeter of the central chamber 21, for instance making 1.5 or 2 or more turns around the perimeter of the central chamber 21. In one embodiment, the elongate channel includes a combination of straight sections and/or curved sections. In one embodiment, the elongate channel 24 includes a constant width. In one embodiment, the elongate channel 24 tapers up to the channel exit port 28. In one embodiment, the elongate channel 24 tapers down to the channel exit port 28. In one embodiment, the elongate channel 24 has a variable width between the channel entrance 21 and the channel exit port 28.
With reference now to
Referring now to
Referring now to
Referring now to
Referring now to
Referring now to
In some embodiments, the assembled HARV device 100 of the present invention comprises face panel disk 110, channel gasket 120, channel disk 130, gas-permeable membrane 140, and back panel disk 150. In some embodiments, the assembled HARV device 100 of the present invention comprises face panel disk 110, channel gasket 120, channel disk 130, a second channel gasket 120, gas-permeable membrane 140, and back panel disk 150. HARV device 100 is assembled by aligning assembly holes 112, 122, 132, 142, and 152 and in some embodiments, introducing a plurality of receiving assembly couplers such as screws, pegs, rods, nails, dowels, and the like through each of the assembly holes of device 100 such that a single assembly coupler extends through each of the aligned assembly holes of each component as described herein thereby sandwiching each layer of device 100 together. In some embodiments, each component of HARV device 100 comprises the same number of assembly holes such that all of the plurality of assembly holes is aligned and coupled when device 100 is assembled.
In some embodiments, the assembled HARV device of the present invention comprises an aligned first channel gasket 120, channel disk 130, and a second channel gasket 120 sandwiched between a face disk 110 on one end, and a gas permeable membrane 140 and back panel disk 150 on the other end. The central chambers of the first channel gasket 120, channel disk 130, and second channel gasket 120 are aligned thereby forming a combined central chamber comprising significantly cylindrical conformation having a high aspect ratio. Similarly, the curved channels of the first channel gasket 120, channel disk 130, and second channel gasket 120 are aligned thereby forming a combined curved channel through which air bubbles may pass. The channel entrance of the first channel gasket 120, channel disk 130, and second channel gasket 120 are significantly aligned thereby forming an entrance for bubbles to pass from the combined central chamber into the combined curved channel. The channel exit of the first channel gasket 120, channel disk 130, and second channel gasket 120 are significantly aligned thereby forming an exit through which bubbles may be expunged. In one embodiment, with reference now to
Embodiments of the present invention further include a drum-shaped STLV device for culturing cells under simulated microgravity conditions while providing a means for eliminating bubbles that can form during normal operation such devices.
Referring now to
Embodiments of the present invention also provides methods for culturing cells under simulated microgravity conditions using the various embodiments of the HARV device and/or the STLV device as described herein. With reference now to
In one aspect, embodiments of the present invention also include a kit comprising instrumentation to apply simulated microgravity conditions to cells or tissues in culture. In certain embodiments, the kit is a sterile packaged kit. In certain embodiments, the one or more instruments of the simulated microgravity kit are sterile and contained in one or more individual sterile packages within the kit. The sterile simulated microgravity kit contemplated herein is thus immediately ready for simulated microgravity application upon removal of the instruments from their respective packages without the need for pre-operation cleaning, sterilizing, or other processing. In certain aspects, the one or more instruments of the simulated microgravity kit are single-use instruments. For example, in one embodiment, the one or more instruments of the simulated microgravity kit are sterile and disposable. In another embodiment, the one or more instruments of the simulated microgravity kit are repackaged after use, where, in certain embodiments, the one or more instruments may be reprocessed for future use. In one embodiment, the instruments may be provided in one or more blister packaging. Each blister may comprise a plastic container (e.g., PETG) component and a lid (e.g., Tyvek®) component.
In certain embodiments, the simulated microgravity kit comprises at least one HARV or STLV as described herein. In certain embodiments, the simulated microgravity kit may optionally include other instruments, such as and without limitation, one or more fittings, tubing, pumps, rotators, bioreactors, and/or reservoirs. For example, in one embodiment, the simulated microgravity kit comprises one or more fittings, which may be barbed fittings, threaded, slip, compression, flare, flange, crimped, and/or pressed fittings or the like, or a combination thereof. In one embodiment, the fluid kit comprises one or more lengths of tubing, including any size or geometry of tubing which may be necessary for the desired application. In one embodiment, the tubing is composed of Tygon®, nylon, polyethylene, or other suitable material known to one skilled in the art. In one embodiment, the tubing comprises one or more stops for integration into holders of a peristaltic pump. In one embodiment, the simulated microgravity kit comprises one or more rotators or bioreactors. In one embodiment, the one or more rotators or bioreactors are single speed, multi-speed, single station, multiple station rotators or bioreactors, or the like, as known to one skilled in the art. In one embodiment, the simulated microgravity kit may include one or more HARVs or STLVs or culturing wells. In one embodiment, the simulated microgravity kit may include culturing media or culturing media components for admixing. In one embodiment, the simulated microgravity kit may include a biological material, such as a cell or tissue.
In certain embodiments, the simulated microgravity kit is custom-configured with regard to size and geometry appropriate to the well being used. In one embodiment, the kit is configured for a specific size of cylindrical body or flange. In an alternative embodiment, the kit comprises instrumentation that may be necessary for various sizes of vessels.
In certain embodiments, the kit comprises instructional material. Instructional material may include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of any of the HARVs or STLVs or simulated microgravity kit components described herein. The instructional material may also include description of one or more steps to perform any of the methods described herein. The instructional material of the kit may, for example, be affixed to a package which contains one or more instruments which may be necessary for the desired procedure. Alternatively, the instructional material may be shipped separately from the package, or may be available electronically, such as accessible from the Internet, or any downloadable electronic document file format.
Still further, embodiments of the present invention include a method of providing a sterile simulated microgravity kit as described herein. In certain embodiments, the method comprises receiving a request for one or more instruments for use in culturing cells or the like. In certain embodiments, the method comprises a customized request for particular instrumentation. In certain embodiments, the method comprises a request for a standardized kit which would contain the one or more instruments. In certain instances, the method comprises gathering the one or more instruments which were requested. In one embodiment, the method comprises processing the one or more instruments. For example, in one embodiment, the one or more instruments are sterilized. Sterilization of the one or more instruments may be conducted by any suitable method known in the art. In one embodiment, the method comprises packaging the one or more instruments. For example, the one or more instruments may be packaged in one or more sterile packages to form a sterile kit.
In one embodiment, the culture chamber filters bubbles, and is also configured to facilitate media changes, such as adding nutrients. In one embodiment, the advantages to the culture chamber discussed herein facilitate miniaturized bioreactor technology. With reference now to
With conventional CPBs 500 (see e.g.
Embodiments of the DPB system and device described herein replace the conventional mechanism by which change occurs, eliminating the need for a minimum cylinder length, allowing working volumes as low as 10 mL in the bioreactor with 5-10 in reserve. The embodiments take the media exchange system off the central axis such that the main cylinder can be made arbitrarily short. In one embodiment, the device incorporate gates or meshes at the entrance of the channel. In one embodiment, the system enables miniaturization to volumes at 10 mL or below. The cost of media in these systems can run in the tens or even hundreds of thousands of dollars over months of culture. Advantageously, the miniaturization component provides a significant advantage. In one embodiment, an angled rotation for the simulation of gravity conditions between 0 and 1 (i.e. partial gravity).
It should be appreciated that the devices and the simulated microgravity kits described herein may be used for a variety of simulated microgravity procedures including but not limited to culturing cells, tissue, biological samples, non-biological samples, and the like. The procedures may be performed on any cell in the human or vertebrate body, including, but not limited to, osteocytes, osteoblasts, osteoclasts, endothelial cells, epithelial cells, smooth muscle cells, mesenchymal cells, progenitor cells, and the like. It should be understood that the present disclosure is not limited to a specific cell, tissue, or simulated microgravity application.
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
In this study, a modification to the traditional RWV was designed and implemented, which resulted in the effective capture and removal of bubbles. Comparison between the modified and standard bioreactors showed similarities in both fluid dynamics and in the generation of organoids under ideal, non-bubble conditions. While the formation of spheroids/organoids was impaired in conventional RWVs in the presence of a bubble, it was fully maintained in the modified design.
Materials and Methods
Bioreactor Design, Modeling, and Computational Fluid Dynamics:
Potential bubble segregating designs were drawn in 2D in AutoCAD 2018 (Autodesk, Mill Valley, Calif.) CAD software. The final design opted for the main body similar to existing RWV bioreactors with an additional thin, exterior, concentric channel running along approximately 80% of the total perimeter, connected to the main body by a thin entrance (
Computational Fluid Dynamics (CFD) was employed to numerically evaluate the fluid dynamics of the new design. The “Fluent” software package in the ANSYS 19.1 release (Ansys, Inc., Canonsburg, Pa.) was used to model fluid behavior in both the novel design and a design emulating the existing 10 ml HARV model in a 2D environment. The simulations were run at 1 G, in water, and at a rotational speed of 10 rpm. The simulations provided the velocity vectors of the fluid at multiple points.
Bioreactor Fabrication:
The bubble capturing bioreactors were designed to be leak-proof, noncytotoxic, sterilizable, and gas permeable for cellular respiration/pH control. Additionally, separate “control” RWVs using identical materials were constructed emulating existing HARV-type bioreactors. The two designs are referred to as “novel” and “standard”, respectively (
All systems were primarily constructed from 3/16″ cast acrylic ACRYLITE® (Evonik Industries, Essen, Germany) and cut on a VLS 6.60 laser cutting table (Universal Laser Systems, Scottsdale, Ariz.). There were three primary pieces for each bioreactor (
Alginate Bead Fluid Dynamics Validation:
To validate the predicted motion of spheroids in the bioreactors, calcium alginate beads (approximately 2 mm in diameter) were used that were produced by dropwise adding a mixture 0.75% sodium alginate (Sigma-Aldrich, St. Louis, Mo.), 1% polyethylene oxide (Sigma-Aldrich, St. Louis, Mo.), and 2% powdered charcoal (for color) into a stirred 2% calcium chloride bath. Approximately two dozen beads were loaded into both types of bioreactors with and without added air bubbles to replicate ideal and failure conditions and rotated at 25 rpm until each system had achieved stable fluid dynamics, as inferred from reduced motion relative to any fixed position on the device. Motion and velocities of beads at the exterior perimeter of the systems were tracked, essentially as previously described (Botta, Manley, Miller, & Lelkes, 2006), using tripod-mounted GoPro camera (GoPro, Inc., San Mateo, Calif.) at a rate of 60 frames per second. The captured images were subsequently evaluated using ImageJ Manual Tracking relative to bubbles and fixed locations on the bioreactor over the course of 1 second. Shear in the absence of bubbles was calculated according to a previously published formula (Ramirez, Lim, Coimbra, & Kobayashi, 2003), describing fluid shear on small particles in an RWV bioreactor, as shown in Equation 1,
τ=μ*|VF−VP|/a Equation 1
Where τ is fluid shear stress, μ is the dynamic viscosity of water a 25° C., VF is the calculated fluid velocity of water at the particle's radius if the fluid were acting as a solid body, VP is the observed velocity of the particle, and a is the radius of the particle (Ramirez, Lim, Coimbra, & Kobayashi, 2003). Because no theoretical velocity can be assumed at the bubble, the traditional fluid shear formula, Equation 2, was used when a particle interacted with a bubble,
τ=μ*δx/δy Equation 2
where δχ is the change in distance (velocity) and δγ is the difference in location (Ramirez et al., 2003). Thus, δχ/δγ is the velocity gradient developed in the fluid between the wall and the particle.
Equations 1 and 2 are substantial simplifications of actual shear equations which are presented as a complex 3D Navier-Stokes equation, the governing equation of motion in fluids (Begley & Kleis, 2000; Munson, Okiishi, Huebsch, & Rothmayer, 2012; Wolf & Schwarz, 1991). All shear values determined from the above equations are reported in dynes/cm2.
Cell Culture for Morphological Assessment:
To perform cell culture, a new sterilization and conditioning protocol was developed and tested. Since the selected materials are not autoclavable and are prone to degradation upon sterilization by 70% ethanol, the protocol was modified from an existing HARV protocol (Botta, Manley, Miller, & Lelkes, 2006). In brief, each bioreactor was filled completely with 0.2 N NaOH for 24 hours. All bioreactors were then moved to a laminar flow hood and exposed to UV sterilization for 30 minutes on each side. They were then emptied of NaOH and rinsed three times with sterile 1×PBS (Corning, Corning, N.Y.). Next, the bioreactors were filled with a sterile conditioning solution composed of 10% FBS (Gibco, Gaithersburg, Md.), 88% lx PBS (Corning, Corning, N.Y.), and 2% Penicillin Streptomycin (Pen-Strep, Gibco, Gaithersburg, Md.) and incubated at 37° C. for 24 hours while rotating at 10 rpm on a 4RCCS rotary system (Synthecon, Inc., Houston, Tex.). After conditioning, bioreactors were emptied aseptically and immediately used for cell culture.
To assess the morphology of the cell-based organoids/spheroids generated in the RWV under ideal conditions, A549 human lung cancer cells (ATCC, Manassas, Va.) were paradigmatically used that have been described extensively in tumor spheroid publications (Maruhashi et al., 2018; Sambale et al., 2015; Zuchowska, Jastrzebska, Chudy, Dybko, & Brzozka, 2017). The cells were maintained in a T75 flask (Falcon) in DMEM (Gibco) supplemented with 10% FBS, 1% Pen-Strep, and 1% L-glutamine (Gibco). Once the cultures reached ˜80-90% confluence, cells were rinsed with PBS and detached using 1× Trypsin (Gibco) and resuspended in DMEM at a concentration of approximately 1×106 cells/ml. Three ml of complete DMEM were added to each bioreactor, followed by 1 ml of the cell suspension. The systems were then completely filled with media using 20 ml syringes (BD, Franklin Lakes, N.J.) and carefully checked to ensure no bubbles were present. For the novel design, the filling was performed through the valve at the terminal end of the channel. Once cells were added, care was taken to ensure the entrance to the channel was oriented vertically to prevent cells incidentally entering the channel. Each bioreactor was then mounted on the 4RCCS system (Synthecon) that was operated at 10 rpm inside a CO2 incubator maintained at 370 C. After 48 hours, the main volume of each bioreactor was collected into 15 ml conical centrifuge tubes (Corning, Corning, N.Y.). Cells and aggregates were sedimented over 5 minutes and then collected with 1000 μL pipettes. Any standard design bioreactor, which exhibited bubble formation, was excluded from these results so that the impact of bubble formation could be tested under more controlled conditions.
In a separate set of experiments, air bubbles were intentionally introduced to both the novel and standard bioreactors prior to culture, in order to evaluate the effects of bubble formation on A549 aggregate formation and spheroid morphology. The above cell culture steps were followed precisely including the complete filling of the bioreactors with media and the elimination of air bubbles. Subsequently, an air-filled 1 ml syringe (BD) was attached to one stopcock while an empty 1 ml syringe was attached to the other. Approximately 250 μL of air was added to each bioreactor to simulate a moderate to severe bubble failure. The bioreactors were then operated under precisely the same conditions as the ideal group. The two groups will be referred to as “without bubble” and “with bubble”, respectively.
Imaging and Evaluation:
For both the “with bubble” and “without bubble” groups, the morphology of the ensuing organoids/spheroids was evaluated by standard image analysis of phasecontrast micrographs. At the end of each culture, the contents of each bioreactor were collected, as described above. Using this method, virtually all aggregates and single cells were collected, which was confirmed by visualizing the supernatant. After sedimentation, 300 μL of spheroid-containing media was collected from the bottom of the tubes and gently transferred to the 14 mm centers of 35 mm glass bottom Petri dishes (MatTek, Ashland, Mass.). At least 10 random images per aliquot were taken of all available spheroids, aggregates, or masses of individual cells at 40× magnification. Images were exported to ImageJ, all identifiable spheroids were manually outlined, and data recorded on each image for area and circularity.
Novel Design Failure Mode Analysis:
One possible limitation of this device is the probability of cells entering the channel prior to the establishment of solid body rotation. To evaluate the probability of this occurrence, Human Dermal Fibroblasts (HDFBs from ATCC) were cultured in complete DMEM (Gibco), as described previously (Lin et al., 2012). One hundred thousand HDFBs were added to each of the bioreactors and incubated on the 4RCCS at 10 rpm for 24 hours. HDFBs were selected due to their tendency not to form aggregates, reducing error due to changing sedimentation rates. After 24 hours, the media in the main volume and the channel volume were collected into separate conical tubes through the main and channel ports respectively, spun down, and resuspended in 1 ml of media. Cell counting was then performed three times on each sample and averages were taken of the totals.
Statistics:
Unless stated otherwise, a minimum of three independent experiments (n=3) were performed for each parameter tested. The calculated areas of the organoids/spheroids are presented as medians±the standard deviation of the medians. Circularity and cell count values are shown as averages±the standard deviation of the medians. Significance was evaluated with a two-tailed student t-test and results were considered significant with a p-value less than 0.05.
Results
Design, Modeling, and Computational Fluid Dynamics:
Bioreactor Fabrication:
Multiple copies of both the standard and novel designs were fabricated; they were leak-proof and functional according to the performance parameters of existing 10 ml HARVtype RWV bioreactors.
Alginate Bead Fluid Dynamics Validation:
Cell Culture:
Shown in
Of note, the volumes of the channels were also collected from the novel design bioreactors. While there were generally little to no aggregates or single cells present, on occasion, a few very large spheroids (of sufficient scale to fall out of suspension, typically >500 μm at the chosen rotational speeds) were found. This may indicate a tendency for spheroids to irreversibly enter the channel, once their sedimentation rates exceed the maximum velocity of the rotating media.
Novel Design Failure Mode Analysis:
Discussion
The aim of this study was to design a modified RWV capable of producing the same low-shear, microgravity-simulating environment as a typical RWV, while capturing and removing nascent bubbles, thus maintaining the critical zero headspace condition. The results in
While the equations used to calculate shear in the alginate bead systems are substantial simplifications, the calculated values, an average ˜0.04 dynes/cm2, under ideal conditions very closely approximated the previously published ideal range of −0.044 dynes/cm2 under zero headspace conditions (Begley & Kleis, 2000). By contrast, in the presence of a bubble (
The prevention of bubble formation is important for the outcome and reproducibility of the results, yet seemingly underreported in the literature (T. G. Hammond & Hammond, 2001). Because bubble formation is frequent, and its prevention/removal is anecdotal, at best, researchers are likely to collect results from RWVs that intermittently formed bubbles (e.g., overnight), not realizing it actually constitutes a flawed experiment. As the use of RWV bioreactor systems is expanding, the presence of bubbles may yield data on unique cellular behavior in these vessels that may, in fact, represent the opposite of what would occur in actual low-shear microenvironement. It is one of our intentions that the dissemination of the novel design may be able to prevent false positive/negative results and increase the reproducibility of the system.
Although the calculated shear stresses and the observed cell spheroid sizes were statistically indistinguishable between the “standard without bubble” group and either of the “novel” groups, there are differences between the standard and novel designs. As described in
Furthermore, in a standard system, any particles interacting with the wall may lose their circular path, negating the modeled microgravity (whole body rotation, zero headspace) environment. However, these cells would still be collected together with the entire bioreactor contents, potentially skewing mechanistic analyses, such as global gene expression profiling in the wake of organoid assembly and differentiation. In the novel system, cells in the main volume would be collected separately from those in the channel volume, ensuring the cells tested are only the ones that did not tend towards the wall, decreasing skew and increasing the consistency of results. Finally, because aggregates grow over time, the tendency for larger aggregates to enter the channel may be leveraged as a mechanism to harvest aggregates of uniform sizes with potential applications in industrial GMP settings.
In summary, a modification to the traditional HARV-type rotating wall vessel bioreactor was engineered and tested, which results in the effective capture and removal of bubbles. The modified and standard bioreactors were compared side by side and found similarities in both fluid dynamics and in the ability to generate cancer spheroids in the absence of bubbles. However, in the presence of a bubble, the spheroid/organoid formation is impaired in conventional RWVs, while it is fully maintained in the modified design. The novel design will increase experimental reproducibility and consistency when using rotating wall vessel bioreactors.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application is a national stage application of PCT Application No. PCT/US18/54937, filed Oct. 9, 2018, and claims priority to U.S. provisional application No. 62/570,177 filed on Oct. 10, 2017, both of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/54937 | 10/9/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62570177 | Oct 2017 | US |
