Cell culture system with altered cell microgravity and shear stress

Abstract

A rotating cell culture system (RCCS) includes: at least one concentric ring attached to a frame along an axis of rotation of the at least one concentric ring; and a DC motor coupled to the at least one concentric ring via one or more gears and configured to rotate the at least one concentric ring along the axis. The RCCS may include at least two concentric rings, where each of the at least two concentric rings is attached to an adjacent ring such that an axis of rotation of at least one concentric ring is orthogonal to an axis of rotation of any other of the concentric rings. The inner ring of the RCCS is configured to hold one or more sample holding members or structures, such as cell culture tubes or microwell plate structures.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 498,105, filed April 25, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0002] This disclosure provides systems and methods for microgravity simulation.

[0003] Long-term space exploration and commercial space travel are becoming increasingly popular as advancements in space technology make journeys safer for everyone, regardless of their profession or training. However, recent observations suggest that more than half of returning astronauts experience infection, colds, or reactivation of dormant viruses within a week of returning to Earth (Rooney BV. FroMicrobiol. 2019; 10:16).

[0004] Furthermore, microgravity can alter cellular processes, including growth and cell organization. Accessing and manipulating cells in these unique cellular states is currently challenging. Developing systems that facilitate the study of cell behavior and processes or that enable cell manipulation under altered microgravity and / or fluid shear forces would be useful.

[0005] Therefore, it is desirable to study the effects of gravity on biological processes, including, for example, regulatory T cell (Treg) activation and seed germination. It is also desirable to investigate the effects on nucleic acid delivery to cells (or cell aggregations) in these unique environments. Summary of the Invention

[0006] This invention provides a multi-axis rotating cell culture system (RCCS) and its uses. In some embodiments, the RCCS may be 3D printed and low-cost. The RCCS can be used to study the activation of regulatory T cells (Tregs) and other biological processes and behaviors in a simulated microgravity (μG) environment. The RCCS can also be used to study the effects of delivering nucleic acids to cells (or cell aggregations) in these unique environments.

[0007] RCCS advantageously enables the study of cell behavior under altered gravity (microgravity) and / or under different fluid shear stresses experienced in cells and / or in various cell culture containers and / or throughout the organism.

[0008] According to one embodiment, a multi-axis rotating cell culture system (RCCS) is provided, comprising up to three concentric rings, wherein the inner ring is configured to hold one or more cell culture tubes, and wherein each ring rotates about an axis orthogonal to the axis of the other rings.

[0009] In some respects, the inner ring is removable and interchangeable.

[0010] Depending on some aspects, the RCCS may also include a DC motor coupled to the outer ring via one or more gears.

[0011] In some respects, the RCCS also includes a speed controller configured to control the operation of the DC motor and the rotation of the three rings.

[0012] According to some respects, the three concentric rings are attached to each other at the points where their opposite axes meet.

[0013] In some respects, the outer ring of the three concentric rings is attached to the frame along the axis of rotation of the outer ring.

[0014] According to some aspects, the RCCS also includes a DC motor coupled to the outer ring via one or more gears and configured to rotate the outer ring along its axis.

[0015] According to one embodiment, a rotating cell culture system (RCCS) is provided, comprising: at least one concentric ring attached to a frame along a rotation axis of the at least one concentric ring; and a DC motor coupled to the at least one concentric ring via one or more gears and configured to rotate the at least one concentric ring along the axis.

[0016] Depending on some aspects, at least one concentric ring is configured to hold one or more cell culture tubes.

[0017] According to some aspects, the RCCS includes at least two concentric rings, wherein each of the at least two concentric rings is attached to an adjacent ring such that the axis of rotation of at least one concentric ring is orthogonal to the axis of rotation of any other concentric ring.

[0018] Depending on some aspects, the inner ring of at least two concentric rings is configured to hold one or more cell culture tubes.

[0019] In some respects, the inner ring is removable and interchangeable.

[0020] According to some aspects, the RCCS consists of three concentric rings.

[0021] According to one embodiment, a method for processing biomaterials in a microgravity environment using a rotating cell culture system (RCCS) is provided.

[0022] According to one embodiment, a method is provided for creating a microgravity environment using a rotating ring system having at least one ring configured to rotate about a first axis. The method typically includes placing a sample within, for example near, the center point or periphery of, at least one ring; and rotating the at least one ring about the first axis.

[0023] In some respects, this method uses any of the multi-axis ring system implementations described herein.

[0024] According to one embodiment, a method is provided for processing biological materials in a microgravity environment using a rotating cell culture system (RCCS) having at least one ring configured to rotate about a first axis. The method typically includes placing a biological sample, for example, near the center point of at least one ring; and rotating the at least one ring about the first axis.

[0025] In some respects, the RCCS also includes a second ring configured to rotate about a second axis orthogonal to the first axis, wherein the diameter of the second ring is greater than the diameter of the first ring, and wherein the first ring is connected to the second ring at a point of connection between the opposing axes, wherein rotating at least one ring includes rotating the second ring.

[0026] In some aspects, the RCCS also includes a second ring and a third ring configured to rotate about a second axis and a third axis, respectively, wherein the second axis is orthogonal to both the first axis and the second axis, wherein the diameter of the third ring is greater than the diameter of the first ring and the diameter of the second ring, and wherein the diameter of the second ring is greater than the diameter of the first ring, and wherein the first ring is connected to the second ring at a point of connection between opposite axes, wherein the second ring is connected to the third ring at a point of connection between opposite axes, wherein the third ring is connected to the frame at a point of connection between opposite axes, and wherein rotating at least one ring includes rotating the third ring.

[0027] In some respects, biological samples include seeds, plant material, or a combination of seeds and a nutrient-rich substrate.

[0028] In some respects, the RCCS also includes a sample holding structure located near the center of the inner ring and held in place by a structure coupled to or integrated into the inner ring.

[0029] In some respects, the inner ring of at least two concentric rings is configured to hold the sample holding structure near the center of the inner ring.

[0030] In some respects, sample retention structures include microplate structures.

[0031] In some respects, the RCCS also includes a bracket with adjustable connector elements, which is configured to change at least one concentric angle to achieve a range of different gravity environments.

[0032] Other features and advantages of the invention will be realized by referring to the remainder of the specification, including the drawings and claims. Other features and advantages of the invention, as well as the structure and operation of various embodiments of the invention, will be described in detail below with reference to the accompanying drawings. In the drawings, the same reference numerals denote the same or functionally similar elements.

[0033] Brief description of several views in the accompanying drawings Figure 1A CAD schematic diagrams of two different orientations of the RCCS implementation scheme are shown.

[0034] Figure 1B A single-loop RCCS according to the implementation scheme is shown.

[0035] Figure 1C A dual-ring RCCS according to an implementation scheme is shown.

[0036] Figure 1D The three-ring RCCS according to the implementation scheme is shown.

[0037] Figure 1E It is a schematic diagram that incorporates the 3D Pythagoras theorem used to calculate the x, y, and z dimensions of gravity.

[0038] Figure 1F A Bluetooth-enabled wireless accelerometer is shown attached to the origin.

[0039] Figure 1G , Figure 1H and Figure 1I A graph showing the g measured in the x, y, and z directions is presented to demonstrate the invalidity of the gravity vector.

[0040] Figure 1J An implementation scheme with three concentric rings is shown.

[0041] Figure 2 A is a schematic diagram of a two-ring device including a 3D-printed polymer transport belt according to the implementation scheme.

[0042] Figure 2 Image B shows a 3D printing and assembly system with a dual-tube retainer as the central ring.

[0043] Figure 2 C shows the vector position data in the x, y, and z directions.

[0044] Figure 2 D shows the average value g obtained by the accelerometer over 2 hours.

[0045] Figure 2F shows accelerometer data measured at various RPM speeds in a 2-ring gear system, where the accelerometers are attached at distances of 0, 0.5, 1.5, 3, or 6 cm from the origin along an axis at a 90° angle to the attachment point. Figure 2 (The yellow line in E).

[0046] Figure 2 F shows the synthesis acceleration of three independent experiments run at 10 RPM for 15 minutes.

[0047] Figure 2 G shows a representative curve of gravity cancellation.

[0048] Figure 2 H shows the synthesis accelerations for three independent experiments run at 1, 3, 10, 25, or 45 RPM.

[0049] Figure 2 Figure I shows a graphical representation of the acceleration in the x, y, and z directions at 45 RPM. This data indicates that these devices can advantageously maintain microgravity at relatively low speeds, ranging from approximately 0.1 g to 6 cm from the origin.

[0050] Figure 3 AG explains the comparison between its implementation plan and commercially available technologies.

[0051] Figure 4 A, 4B, and 4C illustrate interchangeable inner ring design implementations for general dynamic cell culture systems.

[0052] Figure 4 D, 4E, and 4F illustrate the use of attachments respectively on Figure 4 Vector position data measured by accelerometers at the indicated red dots on devices A, 4B, and 4C.

[0053] Figure 5 A-5H shows the successful aggregation and transfection of β-cell aggregates using the implementation scheme (“RPM”), and a comparison with current techniques (Aggrewell aggregation and orbital shaking transfection).

[0054] Figure 6 A is a schematic diagram of cell activation studies conducted under static gravity conditions and with oxygen control conditions in a microgravity device implementation scheme.

[0055] Figure 6 B shows a comparison of CD69 expression in T cells stimulated under microgravity conditions under static gravity and oxygen-controlled conditions.

[0056] Figure 7 Images are shown of different configurations of the microgravity device using different support configurations according to different implementation schemes.

[0057] Figure 8 The design of an inner ring with a sample holding structure (e.g., a plate or block holder) integrated into the inner ring is shown according to an embodiment.

[0058] Figure 9 The diagram shows design schematics of various possible structural modifications to aid stability.

[0059] Figure 10 This study demonstrates various aspects of the biological evidence for the concept, showing that seed treatment in microgravity enhances crop biomass and root growth across species. Detailed Implementation

[0060] According to the implementation plan, a novel, low-cost, multi-axis rotating cell culture system (RCCS) is provided. In some implementation plans, the device may be fully or partially 3D printed.

[0061] Figure 1A A CAD schematic diagram of the device implementation shown in two different orientations is presented; the tandem rings are connected to each other, shown here at 90°, thereby allowing random rotation around the attachment pivot point, where the x, y, and z vector components are indicated at various device rotation positions.

[0062] In implementation schemes, the RCCS design may include one, two, or three rings attached to the opposite axes to allow 360° rotation about the center of mass, such as... Figure 1B , 1C As shown in 1D. More rings can be used. For example, Figure 1B A single-ring embodiment is shown, wherein a single ring 10 is coupled to a motor (e.g., a DC motor) configured to drive the ring 10 (e.g., via a belt or other mechanism) to rotate about its axis. This axis is defined based on connection points 2 and 3 that connect the ring 10 to the frame 5. The connection points to the frame may include pins or other mechanisms that allow the ring to rotate about its axis defined by the connection points, i.e., allow the ring 10 to be rotatably attached to the frame 5. In one embodiment, the axes of each of the outer ring 10 and the inner ring 20 are orthogonal to each other. Similarly, Figure 1C A dual-ring embodiment is shown, wherein an outer ring 10 is coupled to a motor (e.g., a DC motor) configured to drive the outer ring 10 (e.g., via a belt or other mechanism) to rotate about its axis. An inner ring 20 rotates about its axis, which is defined based on connection points 6 and 7 with the outer ring 10. The connection points between the rings may include pins or other mechanisms that allow the rings to rotate about their axes defined by the connection points, i.e., that the attached rings are rotatably attached to each other. Similarly, Figure 1DA three-ring embodiment is shown, wherein the outer ring 10 is coupled to a motor (e.g., a DC motor) configured to drive the outer ring 10 (e.g., via a belt or other mechanism) to rotate about its axis. The inner ring 20 rotates about its axis, which is defined based on connection points 6 and 7 with the intermediate ring 30. The intermediate ring 30 rotates about its axis, which is defined based on connection points 8 and 9 with the outer ring 10 (see...). Figure 1J (This is a limitation.) In one embodiment, the axes of each of the outer ring 10, inner ring 20, and intermediate ring 30 are orthogonal to each other.

[0063] In some implementations, a pinion gear is coupled to a DC motor, which drives a larger gear attached to the outer ring 10 to rotate about the θ axis. In this example, a controller (e.g., an Arduino controller) including a processor and associated memory and communication interfaces is attached to the motor connected to the outermost ring 10. The center or inner ring in each of these devices can be designed to support desired components, such as an accelerometer or one or more tubes. For example, Figure 1B , 1C Images 1 and 2D respectively show an apparatus configured to hold two containers (e.g., 15ml Falcon tubes). The rotational speed of the outer ring 10 can be adjusted by a speed controller, allowing gravity to counteract over time, for example, through centrifugal force and the Coriolis force acting on the unit within the inner ring 20. The speed controller includes a processor and an associated memory that stores instructions to control the DC motor automatically or in response to received control signals. Note that in the single-ring embodiment, the outer ring 10 is the same as the inner ring, therefore "10" and "20" can be used interchangeably in the single-ring embodiment to refer to the "inner ring".

[0064] Figure 1E It is a schematic diagram of the 3D Pythagorean theorem that incorporates the x, y, and z dimensions used to calculate the gravity in the results. Figure 1F A schematic diagram of a Bluetooth-enabled wireless accelerometer attached to the origin of the center or inner ring 20 of devices in rings 1, 2, and 3 is shown. In the study, the device was allowed to rotate randomly, with the motor input set to 10 PRM for 15 minutes. The vector components of acceleration on each axis were measured, and the resulting average gravitational vector was calculated. Figure 1G , 1H Figure 1I shows graphs of g measured in the x, y, and z directions to demonstrate the invalidity of the gravity vector. Each graph includes all data points collected during a 15-minute run from a representative experiment at 10 RPM for each of the 1, 2, and 3-ring systems.

[0065] Figure 1JAn embodiment with three concentric rings is shown. In embodiments with one, two, or three rings, the outer ring 10 is coupled or attached to the frame via a connection point along its axis of rotation, and a DC motor is coupled to the outer ring 10 via one or more gears and configured to rotate the outer ring 10 along its axis of rotation. As shown, each of the three concentric rings is attached to an adjacent ring at opposite axial ends, and each ring is capable of rotating about an axis orthogonal to the axis of the adjacent ring. Furthermore, the inner ring 20 is configured to hold one or more cell culture tubes. In a single-ring embodiment, the single ring is both the inner and outer rings. The axis of rotation of the outer ring is always fixed relative to the frame. In a two-ring embodiment, the two rings rotate along orthogonal axes. In a three-ring embodiment, the axis of rotation of the inner ring 20 may sometimes be parallel to the axis of rotation of the outer ring 10, or may have a component parallel to the axis of rotation of the outer ring.

[0066] Figure 2 A is a schematic diagram of a two-ring device including a 3D-printed polymer conveyor belt according to an embodiment. As shown, the tandem rings are connected to a conveyor belt that connects the outer ring to the inner ring. The rings shown here are connected to each other at 90° using a 1:1 transmission ratio from the motor to the center ring. The conveyor belt in this example is 3D printed from thermoset filaments, which facilitates the thermal recovery of the conveyor belt, allowing it to return to its optimal design shape in the event of wear or stretching. Figure 2 Image B shows a 3D printing and assembly system with dual 15ml Falcon tube retainers as the central ring. Figure 2 C shows the vector position data in the x, y, and z directions measured over 5 minutes, with the accelerometer attached to the central origin of the device running at 10 RPM for up to 2 hours; Figure 2 D shows the average value g obtained by the accelerometer over a period of up to 2 hours. Figure 2 F shows accelerometer data measured at various RPM speeds in a 2-ring gear transmission system, where the accelerometers are attached at distances of 0, 0.5, 1.5, 3, or 6 cm from the origin along an axis at a 90° angle to the attachment point. Figure 2 (The yellow line in E). Figure 2 F shows the synthesis acceleration of three independent experiments run at 10 RPM for 15 minutes, and Figure 2 G shows a representative curve when gravity is ineffective. Figure 2 H shows the synthesis accelerations for three independent experiments run at 1, 3, 10, 25, or 45 RPM, and Figure 2Figure I shows a graphical representation of the acceleration in the x, y, and z directions at 45 RPM. This data indicates that this embodiment of the device can advantageously maintain microgravity of approximately 0.1 g to 6 cm from the origin at lower speeds. (Additional data from other embodiments discussed below show gravity <0.05 G (~5% Earth G).)

[0067] Figure 3 AG illustrates a comparison of implementation schemes with commercially available technologies, wherein vector position data are measured using an accelerometer attached at the sample origin to a 1-ring random 3D-printed RPM (FIG.3A) with a dual-Falcon tube central ring, or using a commercially available RPM (10 RPM) (FIG.3B) set to clinostat mode, or a 2-ring gear 3D-printed RPM (10 RPM) (FIG.3C and FIG.3E), or a commercially available RPM (FIG.3D and FIG.3F) set to 0G mode (2.5 RPM) on the sample stage. Figure 3 AD displays all data points collected in the first 5 minutes. Figure 3 E and F show the data collected 1 hour later. Figure 3 G shows the results of a comparison between a commercially available RPM set to 0G mode and a 3D-printed 2-ring system implementation, measured at various time points up to 60 minutes.

[0068] In one implementation, the inner rings are also interchangeable and can be used to test a variety of cellular environmental conditions, and subsequently test different cell culture flasks with different culture medium volumes to determine how shear stress affects cells in a microgravity-like environment. Figure 4 A, 4B, and 4C illustrate interchangeable inner ring design embodiments for a universal dynamic cell culture system. In these embodiments, the inner ring can be designed to hold any number and size of tubes in various configurations. For example, according to one embodiment, the inner ring can be designed to hold, for example... Figure 4 The two 15mL Falcon tubes shown in B, as... Figure 4 A 50mL Falcon tube as shown in C, or as... Figure 4 A shows up to fourteen 1.5 mL centrifuges.

[0069] Figure 4 D, 4E, and 4F illustrate the use of attachments respectively on Figure 4 Vector position data measured by accelerometers at the indicated red dots on devices A, 4B, and 4C. All devices are operating at 10 RPM. Figure 4 DF shows all the data points collected in the first 15 minutes, and the average effective gravity at 15 minutes.

[0070] Biological Evidence from Conceptual Research: Aggregation and Transfection The preparation of cell aggregates is often challenging, and transfection strategies for transfecting whole aggregates or organoids are limited. Figure 5 A-5H shows the successful aggregation and transfection of β-cell aggregates using the implementation scheme (“RPM”), and a comparison with current techniques (Aggrewell aggregation and orbital shaking transfection). Figure 5 AC shows the aggregates formed after overnight shaking (scale bar 320 μm). Figure 5 DH showed cells treated with 3500 ng / ml eGFP mRNA in lipid nanoparticles, and transfection was analyzed by confocal microscopy and flow cytometry. Figure 5 In DF, the scale bar is 100 μm. The 3D printing random positioning machine of the implementation scheme is superior to other systems used for combinatorial polymerization and transfection.

[0071] Biological evidence from conceptual studies: Immune cell stimulation after exposure to microgravity Figure 6 A is a schematic diagram of cell activation studies conducted under static gravity conditions and with oxygen control conditions in a microgravity device implementation scheme. Figure 6 Figure B shows a comparison of CD69 expression in T cells stimulated under microgravity conditions versus under oxygen-controlled conditions. In microgravity, T cells showed reduced activation, indicating a more immunosuppressive phenotype. This data is similar to data already published in other microgravity systems, validating the device implementation. It also suggests that the device could be used for the expansion of modulatory or immunosuppressive cell types.

[0072] In the implementation plan, the effective G experienced can be modified or controlled using different support structures depending on the implementation plan. Figure 7 Images of different structures of the microgravity device using different support structures according to the implementation scheme are shown. The addition of the support alters the effective G experienced by the sample within the ring. Figure 7 In the diagram, small figure A shows the normal (45°) support structure, small figure B shows the modified structure with the motor facing downwards (e.g., 0°), and small figure C shows the modified structure with the motor facing upwards (e.g., 90°). The modified support facilitates changes in the angle of the rings within the device, which is beneficial for achieving even lower G values. Figure 7 In the middle, small figure D shows the allowed... Figure 2 B and Figure 2 CAD image of an angled track for an example structure in C. Figure 7The small graph EH shows a graphical representation of the composite acceleration with a normal (45°) and modified (90°) pedestal. Data were collected using a WitMo accelerometer placed between two 15 mL Falcon tubes within the 2-loop system, and then organized and analyzed using Excel and Python. The device was run at 10 and 15 RPM for at least 30 minutes with each pedestal. All graphical representations shown here exclude data from the first 5 minutes of each run, during which time the device begins to accelerate and its motion becomes smoother. The motor used with the current device is a 12V (max) 60 RPM motor. Figure 7 In the figure, small figure E shows the operation at 10 RPM, where a standard (45°) support was used from t=5 minutes to t=60 minutes; the calculated average g was 0.038 g. Figure 7 In the figure, small figure F shows the operation at 15 RPM, where the normal (45°) stent was in place from t=5 minutes to t=30 minutes; the calculated average g was 0.301 g. Figure 7 In the figure, small figure G shows the operation at 10 RPM, where the modified (90°) support was used from t=5 minutes to t=60 minutes; the calculated average g was 0.027 g. Figure 7 In the figure, small figure H shows the operation at 15 RPM, where the modified (90°) support was used from t=5 minutes to t=60 minutes; the calculated average g was 0.019 g. 0.019 G is approximately 2% of the Earth's normal gravity.

[0073] Figure 8 The design of an inner ring 20 with a sample holding structure 22 (e.g., a plate or block holder) integrated into the inner ring is shown according to an embodiment. In embodiments, the sample holding structure may include one or more components. For example, in the illustrated embodiment, each of the three components may be printed separately or otherwise formed and then assembled via a convex-concave connector on each side ring and the center feature. The holding structure 22 is positioned within a protruding segment around the center of each arc. Due to the weight distribution of the rectangular container, the four support points should offset any differences. The rectangle in the above image represents the container / holding structure 22 (e.g., a microporous structure, such as a 96-well plate with dimensions of 12.5 cm × 8 cm × 1.5 cm). Ring 23 represents the center ring, where arc 24 depicts the peripheral components that will be added once the plate has been centered. In the illustrated embodiment, the entire structure is designed to be compatible with current 2-ring systems and should be able to be quickly interchanged with the inner ring, but it can also be used with one-ring and three-ring embodiments. Figure 8 In the diagram, small figure A shows the separate components, and small figure B shows the combination of all three components along the male-female connector.

[0074] Figure 9The diagram shows design schematics of various possible structural modifications to aid stability.

[0075] Figure 10 This study illustrates various aspects of the biological evidence for a concept study demonstrating that seed treatment in microgravity enhances crop biomass and root growth across species. Seeds were grown on a nutrient-rich substrate (e.g., a nutrient-soaked fiber pad) and exposed to simulated microgravity (~0.03 G, 18 hours 10 RPM) within a device under ambient light conditions. The seeds were then transferred to the fiber pad and allowed to grow for an additional 48 hours under normal Earth gravity conditions before harvesting. Seedlings were imaged and weighed. Characteristics including root length, seedling length, number of roots, root branching, and number of leaves were recorded. Inset A shows a schematic diagram of the growth protocol. Inset B shows seeds embedded in a fiber pad for microgravity stimulation. Figure C shows a bright-field image of mixed microgreens taken 48 hours after an 18-hour germination stage in standard Earth gravity or simulated microgravity. Figure D shows bright-field and fluorescence images of roots of Brassica var. nipposinica seedlings grown for 18 hours in normal or simulated microgravity before planting. Images were taken 48 hours after returning to Earth's normal gravity. Roots were stained with propidium iodide. Scale bar: 500 micrometers. Small figure EN shows the various edible crops studied: a mixture of microgravity seeds [a mixture of *Brassica oleracea* var. *palmifolia*, *Brassica rapa* subsp. *chinensis*, *Brassica rapa* var. *nipposinica*, and *Brassica oleracea* var. *capitata* f. *rubra*] as well as *Lepidium sativum*, *Brassica rapa* chinensis, *Brassica rapa* var. *nipposinica*, and *Brassica oleracea* Lacinato, which were treated and analyzed separately as described. Error bars are SEM images. Approximately 30–60 seeds were tested under each condition. In summary, seed exposure to microgravity within the apparatus before planting significantly increased root length and root branching in some species. This increase in biomass should be useful for stabilizing plants in different soil topography and could increase carbon capture by plants without compromising crop yield.

[0076] Use Cases: The frequency and duration of space exploration and commercial space travel are increasing. However, the tools available for studying cellular changes during space travel, particularly in the microenvironment of space, are limited, leading to the development of technologies to protect astronauts from harm during space travel. For example, one currently available technology for studying the effects of microgravity on cell behavior is the single-axis rotating cell culture system (RCCS). This system does not allow cells to move along multiple axes, is limited to the cell culture flasks that can be studied, and does not allow for the simultaneous control or study of other important characteristics such as fluid flow rate, shear stress, etc. This implementation offers several novel advancements and new features compared to currently commercially available single-axis RCCS systems and can be used in other applications.

[0077] Appendix A of U.S. Provisional Application No. 63 / 498,105 provides additional aspects and features of the implementation of the RCCS. Appendix A is incorporated herein by reference. All references cited herein, including publications, patent applications and patents, are incorporated herein by reference to the extent that each reference is individually and specifically indicated as incorporated by reference and fully set forth herein.

[0078] In the context of describing the disclosed subject matter (particularly in the context of the following claims), the use of the terms “a,” “an,” “the,” and “at least one,” and similar indicators, should be interpreted as covering both the singular and the plural, unless otherwise stated herein or obviously contradicted by the context. The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted as referring to one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise stated herein or obviously contradicted by the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, the description of value ranges herein is intended only as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually described herein. All methods described herein may be performed in any suitable order, unless otherwise stated herein or obviously contradicted by the context. The use of any and all examples or example language (e.g., "such as") provided herein is intended only to better illustrate the disclosed subject matter and does not constitute a limitation on the scope of the invention unless otherwise required. The language in the specification should not be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0079] This document describes certain embodiments. Variations of these embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the embodiments in ways different from those specifically described herein. Therefore, this disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, this disclosure covers any combination of the foregoing elements in all possible variations.

Claims

1. A multi-axis rotating cell culture system (RCCS) comprising up to three concentric rings, wherein the inner ring is configured to hold one or more cell culture tubes, and wherein each ring rotates about an axis orthogonal to the axes of the other rings.

2. The RCCS of claim 1, wherein the inner ring is removable and interchangeable.

3. The RCCS of claim 1 further includes a DC motor coupled to the outer ring via one or more gears.

4. The RCCS of claim 3 further includes a speed controller configured to control the operation of the DC motor and the rotation of the three rings.

5. The RCCS of claim 1, wherein the up to three concentric rings are attached to each other at opposite axis connection points.

6. The RCCS of claim 1, wherein the outer ring of the up to three concentric rings is attached to the frame along the rotation axis of the outer ring.

7. The RCCS of claim 6 further includes a DC motor coupled to the outer ring via one or more gears and configured to rotate the outer ring along its axis.

8. A rotating cell culture system (RCCS) comprising: At least one concentric ring and a DC motor, the at least one concentric ring being attached to a frame along a rotation axis of the at least one concentric ring, the DC motor being coupled to the at least one concentric ring via one or more gears and configured to rotate the at least one concentric ring along the axis.

9. The RCCS of claim 8, wherein the at least one concentric ring is configured to hold one or more cell culture tubes.

10. The RCCS of claim 8, comprising at least two concentric rings, wherein each of the at least two concentric rings is attached to an adjacent ring such that the axis of rotation of the at least one concentric ring is orthogonal to the axis of rotation of any other concentric ring among the concentric rings.

11. The RCCS of claim 10, wherein the inner ring of the at least two concentric rings is configured to hold one or more cell culture tubes.

12. The RCCS of claim 11, wherein the inner ring is removable and interchangeable.

13. The RCCS according to claim 11, comprising three concentric rings.

14. A method for processing biomaterials in a microgravity environment using the rotating cell culture system (RCCS) according to claim 8.

15. A method for processing biological material in a microgravity environment using a rotating cell culture system (RCCS), said rotating cell culture system (RCCS) having at least one ring configured to rotate about a first axis, the method comprising: Place the biological sample within at least one of the loops; as well as The at least one ring is rotated about the first axis.

16. The method of claim 15, wherein the placement comprises placing the biological sample near the center point of the at least one ring or near the periphery of the at least one ring.

17. The method of claim 15, wherein the RCCS further comprises a second ring configured to rotate about a second axis orthogonal to the first axis, wherein the diameter of the second ring is greater than the diameter of the first ring, and wherein the first ring is connected to the second ring at a point of connection with the opposite axis, wherein rotating the at least one ring includes rotating the second ring.

18. The method of claim 15, wherein the RCCS further comprises a second ring and a third ring configured to rotate about a second axis and a third axis, respectively, wherein the second axis is orthogonal to both the first axis and the second axis, wherein the diameter of the third ring is greater than the diameter of the first ring and the diameter of the second ring, and wherein the diameter of the second ring is greater than the diameter of the first ring, and wherein the first ring is connected to the second ring at a point of connection between opposite axes, wherein the second ring is connected to the third ring at a point of connection between opposite axes, wherein the third ring is connected to the frame at a point of connection between opposite axes, and wherein rotating the at least one ring includes rotating the third ring.

19. The method of claim 15, wherein the biological sample comprises a seed, plant material, or a seed and a nutrient-rich substrate.

20. The RCCS of claim 1 further includes a sample holding structure located near the center of the inner ring and held by a structure coupled to or integrated into the inner ring.

21. The RCCS of claim 10, wherein the inner ring of the at least two concentric rings is configured to hold the sample holding structure near the center of the inner ring.

22. The RCCS of claim 21, wherein the sample holding structure comprises a microporous plate structure.

23. The RCCS of claim 8, further comprising an adjustable bracket having connector elements, the bracket being configured to change the angle of the at least one concentricity.

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