Patent Application
20220186168
The presently disclosed subject matter relates generally to devices for biologic studies. Particularly, the presently disclosed subject matter relates to rotating suspension culture devices that allow direct microscopy, in situ assays, and automation.
The present application is related to U.S. Design Patent Application titled SUSPENSION CULTURE DEVICE and another U.S. Design Patent Application titled SUSPENSION CULTURE DEVICE, each filed simultaneously herewith.
Cells in the body are exposed to flow shear as fluids, such as blood or renal glomerular filtrate, flow past the outer cell membrane. In the kidney, fluid from the blood is filtered in the glomerulus and this ultrafiltrate flows past proximal tubule cells (PTC) that are responsible for reabsorbing water, sodium, glucose, amino acids, and diverse hormones and proteins. This is not a languid process—the kidney generates over 100 ml of ultrafiltrate per minute and the proximal tubule cells are responsible for reabsorbing 70% of this volume. Calculations of the fluid shear stress in vivo is complicated by the varying dimensions of the tubules and varying composition of the ultrafiltrate as it moves down the tubule, but it is estimated that PTC are exposed to shear stress in the range of 0.04-2 dynes/cm2. Higher level of fluid shear stress can occur during renal dysfunction and are thought to contribute to disease progression and play a key role in progression of chronic renal disease.
Fluid shear stresses have an important role in maintaining the differentiation of PTC. To be meaningful and representative models of living kidneys, cultured proximal tubule cells must be exposed to fluid shear stress in vitro. Exposure to fluid shear stress increases PTC transport of proteins, expression of microvilli, and formation of tight junctions with increased transepithelial electrical resistance.
Several limitations may account for discrepancies between the many published reports on the effects of flow shear stress on cultured PTC. Some investigators use primary PTCs from human, rat, or mouse kidneys. Others use cell lines such as H2K, MDCK, RPTEC/TERT1, OK opossum kidney cell line, or SV40 transformed proximal tubular epithelial cells (PTEC), each of which varies as to how well they maintain the function of in vivo cells. The shear stresses employed in these studied range from 0.02 to 9.0 dynes/cm2 and are applied for times ranging from 15 minutes to over two weeks. Finally, the flow shear stress is applied using diverse technologies including orbital shakers, parallel plates, microfluidics with peristaltic pumps, and perfused hollow fibers. Many of these options require expensive equipment and are rarely practical for evaluating large numbers of replicates due to cost and vessel volume.
Suspension cultures, in which cells float in a liquid milieu, have significant advantages for the delivery of physiologic levels of flow shear stress. Suspension culture technology has been modelled, validated experimentally, and matured for routine use. Roller bottles, paddle stirrers, and shakers are inexpensive options and quite suitable for fungi, bacteria, and algae that can tolerate high shear levels and are relatively resistant to injury from impact against the vessel walls. But mammalian cells need much gentler treatment to avoid cellular damage and to mimic the shear levels they experienced in vivo.
Rotating suspension cultures can provide physiologic levels of fluid shear stress. Controlled shear is achieved by zero head space, that is filling the vessel entirely with culture media, so that the contents rotate in laminar flow and avoid turbulent flow entirely. The rotating wall vessel spins around a horizontal axis and the cells move in an annulus around the axis of rotation. Cells and aggregates of different size and density co-localize in the annulus. Cells do not need to adhere to a plastic surface and thereby avoid the de-differentiation associated with 2D cultures. However, cells can be attached to beads or other scaffolds, as needed. A gas permeable membrane allows for gas exchange.
Rotating suspension culture has found limited applicability due to limitations of the currently available hardware. Re-usable vessels have multiple components needing autoclaving at different temperatures, as well as manual assembly in a cell culture hood. The vessels attach to spindle rotators that spin with great precision. However, the rotators are expensive and can only hold a few vessels. Commercial applications are largely limited to generation of large numbers of tissue spheroids that are transferred to other systems for experimentation.
In view of the foregoing, there is a dire need for small suspension culture devices that are affordable, simple to use, and adaptable for use in studies with large numbers of replicates. Further, there is a parallel need for larger affordable, simple to use suspension culture devices to produce commercial quantities of biomolecules.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Drawings, which are not necessarily drawn to scale, and wherein:
The presently disclosed subject matter relates to rotating suspension culture devices that allow direct microscopy, in situ assays, and automation. According to an aspect, a suspension culture device includes a rotatable base having an exterior surface that engages at one or more rollers for rotation of the base about an axis when the at least one roller is turning. The device includes first and second end components attached to the base along the axis. The base and the first and second end components define an interior space for holding liquid. A portion of at least one of the end components is made of a material that is at least partially transparent for viewing into the interior space from outside the base. Further, the device includes ports that each permit fluid communication between the interior space and outside the base.
According to another aspect, a suspension culture system includes one or more rollers. Further, the system includes a mechanism configured to turn the rollers. The system also includes a suspension culture device including a rotatable base having an exterior surface that engages the roller(s) for rotation of the base about an axis when the at least one roller is turning. Further, the system includes first and second end components attached to the base along the axis, wherein the base and the first and second end components define an interior space for holding liquid, wherein a portion of at least one of the end components is made of a material that is at least partially transparent for viewing into the interior space from outside the base. Further, the system includes ports that each permit fluid communication between the interior space and outside the base.
According to another aspect, an adaptor is disclosed for holding a suspension culture device for observation of contents of the suspension culture device. The adaptor includes a base portion comprising a top portion defining a surface and a bottom portion defining a surface. Further, the base portion defines an aperture that extends between the surface of the top portion and the surface of the bottom portion. The adaptor includes a suspension culture device holder comprising a first feature and a second feature. The first feature is configured for holding a suspension culture device. The second feature is configured for fitting to the aperture of the base portion.
The following detailed description is made with reference to the figures. Exemplary embodiments are described to illustrate the disclosure, not to limit its scope, which is defined by the claims. Those of ordinary skill in the art will recognize a number of equivalent variations in the description that follows.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used to provide flexibility to a numerical endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
The use herein of the terms “including,” “comprising,” or “having,” and variations thereof is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting” of those certain elements.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range is stated as between 1%-50%, it is intended that values such as between 2%-40%, 10%-30%, or 1%-3%, etc. are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
In accordance with embodiments of the present disclosure, some or the entirety of the rotatable base 102 may be made of a breathable material that extends between the interior space and outside the rotatable base 102. As a result, oxygen or other gas from outside the rotatable base 102 may enter into the interior space to thereby allow cells in the culture to maintain their metabolism and differentiation. Further, gases such as carbon dioxide, produced by cell metabolis, can escape the interior space. The breathable material is selected for differential gas exchange such that water is retained orders of magnitude better, than oxygen and carbon dioxide are diffused. In some embodiments, the rotatable base 102 has one or more portions that are thinner than other portions to provide an easier pathway for oxygen from the outside into the interior space. These portions of the rotatable base 102 can be any suitable size, shape, and provide any suitable thickness between the outside and the interior space. An example of these portions is 0.001″ thick FEP (fluorinated ethylene propylene). In examples, these portions can take the form of divots, indentations, and the like in the base 102. Moreover, the rotatable base may be designed for controlling an amount of oxygenation, carbon dioxide removal, and water retention, desired within the interior space where the culture is located.
The breathable material of the base 102 may be any suitable material that permits gas to pass through it. Example breathable material includes, but is not limited to, fluoroplastic, fluorinated ethylene propylene (FEP), PerFluoroAlkoxy (PFA), polytetrafluoroetylene (PTFE), the like, and combinations thereof.
The device 100 shown in
In this example, a port 108 is made of a silicone rubber material that is positioned within a hole defined in the base 102. The hole provides a passageway that extends from outside the base 102 to the interior space. A blunt or sharp (sharp needle hole can seal better) needle (e.g., 18 to 26 gauge blunt or sharp needle) or other suitable instrument may penetrate the rubber material of the port 108 such that liquids can be introduced into the interior space. Once the needle is removed, the rubber material may reseal the port 108. Air may be bled from the interior space by use of another needle at another port.
As shown in
The device 102 also includes multiple windows 110 attached to the base 102 for permitting viewing into the interior space. For example, cells in the interior space may be stained with fluorescent dyes and imaged by inverted fluorescent microscopy. In this example, the base 102 defines multiple apertures 112 that lead to where respective windows 110 are positioned. The contents in the interior space may be observed by viewing through an aperture 112 and its respective window 110. The windows 110 are sealed such that fluid cannot escape from the interior space. Further, the windows 110 may be made of transparent, semi-transparent, or substantially transparent such that a person or instrument may see through the window 110 into the interior space. In an example, the windows 110 may be made of FEP and have a thickness of between about 0.0005″ and 0.05″. As in described herein, example breathable material includes, but is not limited to, fluoroplastic, fluorinated ethylene propylene (FEP), PerFluoroAlkoxy (PFA), polytetrafluoroetylene (PTFE), the like, and combinations thereof.
Alternatively, the material of the base 102 may be partially or entirely transparent such that the contents of the interior space can be viewed from the outside.
It is noted that the components may be made by 3D printing or any other suitable technique, such as injection molding. Examples include, but are not limited to, FEP and PFA techniques.
The bottom component 102C also has cantilever snap-fits 500 and can be moved towards the middle component 102B to similarly attach to the middle component 102B on its opposing side. As shown in
As shown in
It is noted that although cantilever snap-fits 500 are used in this example as attaching the components 102A, 102B, and 102C together, it should be understood that any other suitable mechanism may be used for attaching the components 102A, 102B, and 102C together.
Ports 108 each include an aperture 108A and a pliable material 108B that fits into the aperture 108B. The pliable material 108B can be made of silicone rubber and defines a passageway 108C that extends between outside the base 102 to the interior space 506. The passageway 108C may be used for introducing culture into the interior space 506 or removing air, another gas, or liquid from the interior space 506.
With continuing reference to
With continuing reference to
The use of the adaptor 1100 with the device 100 brings the device into the focal length of lenses commonly in use on inverted microscopes.
With continuing reference to
For experimentation, the device 100 of
Simulations were performed to evaluate the fluid mechanical forces experienced by cells in the spinpod.
The viability of RPTEC/TERT1 renal cells in rotating spinpods was not significantly different from that of static spinpods at the end of three days of culture (
The next generation sequencing shows a different sequence and timing of responses of RPTEC/TERT1 renal cells in spinpods when they are static or rotated (Table I below). At 3 hours the cells in static spinpods are already displaying increases in RNA gene expression and RNA polymerase biosynthesis. There are already cellular changes in cytokine signaling, apoptotic cell death, immune effector defense, and intracellular protein phosphorylation. By 24 hours the cells in static spinpods have large changes in oxygen compound response, and apoptotic process regulation. At the same 24-hour time period, the rotating cells are showing changes in cell cycle regulation, apoptosis, and catabolic processes. Again, this is consistent with our flow cytometry and cytokine data. By 72 hours the cells in static spinpods show changes in DNA metabolic response, oxidation reduction processes, oxidative stress response, cell cycle, and lipid metabolism. At the same 72-hour time point the rotating cells demonstrate changes in response to toxic compounds, cell death regulation, and vessel morphogenesis development.
Notably, of all the common, well-characterized renal transporters, the only one that changed in the rotating spinpods was the breast cancer resistance protein (HGNC Gene Symbol ABC-G2, common symbol BCRP). BCRP was reduced at 3 hours in the static cultures (differential expression q-value 0.031), but this reduction was delayed in rotating cultures with differential expression q-values of 0.036 at 24 hours, and 0.02 at 72 hours respectively. There was no change at any time point in other drug transporters known to be expressed by PCT including Organic Anion Transporter 1 (OAT1), Organic Anion Transporter 3, (OAT-3) Organic Anion Transporter 4 (OAT-4), Urate Anion Exchanger 1, Organic Cation Transporter 2 (OCT-2), Multidrug and Toxin Extrusion Protein 1 (MDR-1, also known as MDR-1 and P-gp), Multidrug Resistance Associated Protein 2 (MDR-2), Multidrug Resistance Associated Protein 2 (MDRAP-1), or Multidrug Resistance Associated Protein 4 (MRAP-4).
As the time of exposure increased, RPTEC/TERT1 cells exposed to flow shear stress began to express more and different genes compared to cells cultures under static conditions. Table II below lists the RPTEC/TERT1 genes whose expression was significantly increased or decreased in rotating spinpod cultures compared to static spinpod cultures at the 3 hour, 24 hour, and 72 hour time points.
The quantity of thirteen cytokines/chemokines were measured in the supernatants of RPTEC/TERT1 after 72 hours in rotating and static spinpods: IL1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF, IFNγ, MCP-1, and TNFα. Four of these were present in significantly different quantities in the supernatant of PCT exposed to rotation compared to static cultures (Table III and
While the embodiments have been described in connection with the various embodiments of the various figures, it is to be understood that other similar embodiments may be used, or modifications and additions may be made to the described embodiment for performing the same function without deviating therefrom. Therefore, the disclosed embodiments should not be limited to any single embodiment, but rather should be construed in breadth and scope in accordance with the appended claims.
