CN111770987A

CN111770987A - A support loader for bioreactor

Info

Publication number: CN111770987A
Application number: CN201980015435.4A
Authority: CN
Inventors: Z·王; W·刘; F·佩文迪; A·雅达利
Original assignee: 3D Biotek LLC
Current assignee: 3D Biotek LLC
Priority date: 2018-02-27
Filing date: 2019-02-26
Publication date: 2020-10-13
Also published as: WO2019168838A1

Abstract

A system for seeding cells onto an array of a mesh plate or scaffold is provided. The system may include a storage screen holder and a loadable screen holder which may be in a spaced relationship such that a screen may be moved from the storage screen holder into the loadable screen holder when pushed by the pushing mechanism. The cells for seeding may be dispensed through a dispenser. The motion control system may move any number of the appropriate screens, screen holders and dispensers as desired. The lowest plate can be moved into the loadable plate holder and cells can be dispensed thereon, and then successively higher plates are moved into the loadable plate holder and seeded with the cells. Components such as storage screen holders, loadable screen holders and screens may be provided as pre-sterilized components.

Description

A support loader for bioreactor

This application claims the benefit of U.S. provisional application No. 62/636,039 filed on 27.02/2018, the entire disclosure of which is incorporated herein by reference.

Technical Field

Embodiments of the present invention relate to bioreactors.

Background

Bioreactors are used to expand cell populations, such as stem cells or other anchorage-dependent cells. However, improvements are still needed, for example in terms of ease of use, and automation and program reproducibility (reproducibility procedure). It is desirable to perform the culture to produce a large number of cells, for example, billions of cells if possible. Rack seeding is an area where improvement would be useful. Various bioreactors and their components are described in U.S. non-provisional patent application publication nos. 2018/0057784 and 2019/0002815, the disclosures of which are hereby incorporated by reference in their entirety.

Disclosure of Invention

In an embodiment of the invention, there may be provided a system for creating a stack of cell-seeded mesh plates (screen), the system comprising: a plurality of net plates; a storage screen holder adapted to receive a plurality of screens; a loadable mesh panel holder adapted to receive a plurality of mesh panels; a pusher adapted to push individual ones of the mesh panels from the storage mesh panel holder to the loadable mesh panel holder; a dispenser adapted to dispense a liquid containing the cells onto one of the well plates; wherein the storage screen holder and the loadable screen holder are arranged at a distance from each other such that one of the screens accommodated in the storage screen holder can be slid into the loadable screen holder in a planar translational movement; and a motion control system adapted to control the motion or position of at least some of the storage screen holder, the loadable screen holder, the pusher, the screen and the dispenser.

Embodiments of the invention may include a method for creating a stack of cell-seeded mesh plates, the method comprising the steps of: providing a storage screen holder; providing a plurality of mesh panels, the mesh panels being unsown; loading a plurality of screen plates into a storage screen holder, the screen plates being loaded one on top of the other; providing a loadable mesh panel holder; assembling a storage screen holder and a loadable screen holder to each other to form an assembly configured such that any one screen in the storage screen holder can be slid into a corresponding position in the loadable screen holder; mounting the assembly to a machine comprising a motion control system and a dispenser; and operating the machine to move a lowermost one of the plurality of panels in the storage panel holder into the loadable panel holder as a first moving panel and then to dispense liquid from the dispenser onto the first moving panel, then to move a next higher panel from the storage panel holder into the loadable panel holder and then to dispense liquid from the dispenser onto a next higher panel.

Embodiments of the invention may include a method for creating a cell-seeded mesh plate, the method comprising the steps of: providing a web sheet, the web sheet being unsown, wherein the web sheet comprises four layers, each layer comprising fibers that are substantially straight and substantially parallel to other fibers in the layer, the fibers being generally perpendicular to fibers in adjacent layers of the web sheet, the fibers within a layer being separated from adjacent fibers within the layer by a separation distance greater than a fiber diameter of the fibers, wherein the web sheet has a see-through region (see-through region) having a largest dimension less than the fiber diameter in plan view, wherein the contact angle of the fibers with pure water is less than 50 degrees; providing a plurality of liquid distributors arranged in a regular array, wherein the distributors define a unitary body (unit cell) having a planar area of a mesh panel associated with an individual one of the distributors, wherein the unitary body further has a vertical dimension which is the distance between the top of the mesh panel and the bottom of the mesh panel, wherein the unitary body has a total volume equal to the planar area multiplied by the vertical dimension, wherein the unitary body further has a solid volume occupied by the fibers within the unitary body, wherein the unitary body further has an empty volume, i.e. said total volume minus said solid volume; liquid is dispensed from the dispenser onto the screen in a vertically downward direction substantially perpendicular to the screen, wherein a volume of the dispensed liquid fills 40% to 60% of an open volume of the screen.

Drawings

Embodiments of the present invention are further described, but in no way limited to, the following illustrative examples:

FIG. 1 is a three-dimensional perspective view showing a loadable mesh panel holder without mesh panels;

FIG. 2 is a three-dimensional perspective view showing the loadable mesh-plate holder of FIG. 1 exploded;

FIG. 3A is a three-dimensional perspective view showing a loadable mesh panel holder with a mesh panel therein;

FIG. 3B is a three-dimensional perspective view showing the loadable mesh plate holder of FIG. 3A with a portion of the loadable mesh plate holder missing to better show the mesh plate;

FIG. 3C is a three-dimensional perspective view showing the loadable mesh panel holder of FIG. 3B with some mesh panels missing;

FIG. 4A is a view of a closure of the loadable mesh plate holder, showing a circular entrance (round entry) of a slit in the loadable mesh plate holder;

FIG. 4B is another view of the closure of the loadable mesh plate holder from a different perspective;

FIG. 4C is a view of a partial area of a closure with a mesh panel present;

FIG. 5A shows a mesh panel made according to an embodiment of the present invention;

FIG. 5B shows a closer view of a similar mesh panel;

FIG. 5C illustrates spacing parameters of mesh panels such as those in FIGS. 5A and 5B;

FIG. 5D more particularly illustrates the arrangement of fibers within the web and their staggering;

FIG. 5E shows a plan view of only one layer of a mesh sheet;

FIG. 5F shows a plan view of only two layers of mesh sheet;

FIG. 5G shows a plan view of only three layers of mesh sheets;

FIG. 5H shows a plan view of four layers of a mesh sheet;

FIG. 5I illustrates a mesh sheet having a non-uniform thickness;

FIG. 6 illustrates a possible leakage flow path;

FIG. 7A illustrates a three-sided piece (threaded piece) as part of a loadable mesh plate holder to illustrate the positioning slots;

FIG. 7B is a sectional view (cutaway view) illustrating the three-sided component of FIG. 7A along with a pusher attachment;

FIG. 7C illustrates a pusher attachment;

FIG. 8A is a three dimensional illustration of a storage screen holder;

FIG. 8B is another three-dimensional illustration of a storage screen holder, also showing several screens;

FIG. 9A is a three-dimensional illustration of a storage screen holder and a loadable screen holder placed in a side-by-side position;

FIG. 9B is the cross-section of FIG. 9A, further showing one of the mesh panels;

FIG. 10A is an exploded three-dimensional illustration of a storage screen holder and a loadable screen holder separated from each other for clarity of illustration;

FIG. 10B is a cross-section of FIG. 10A;

FIG. 11A is a three-dimensional illustration of a seeding tray;

FIG. 11B is a three-dimensional illustration showing a storage screen holder and a loadable screen holder side-by-side in a seeding tray supporting and positioning them;

FIG. 12A is a three dimensional view of a storage screen holder and a loadable screen holder and a stop (stop) adapted to prevent movement of the screen during transport;

FIG. 12B is a three-dimensional perspective view of the handle in isolation;

FIG. 12C is a three-dimensional perspective view of the handle in combination with the loadable mesh panel holder;

FIG. 13A is an exploded view illustrating a pusher and its support;

FIG. 13B is similar to FIG. 13A from a different perspective;

FIG. 14A is a three dimensional view of a storage screen holder and loadable plate holder and a dispenser for dispensing cells for seeding onto the screen;

FIG. 14B is similar to FIG. 14A except from a different perspective;

FIG. 14C is a cross-sectional view of FIG. 14B; and

fig. 15 is a three-dimensional perspective elevation view of the entire system or machine.

Detailed Description

Reference is now made to fig. 1 to 7C. As described in U.S. patent application No. 15/686,211, in an embodiment of the present invention, a bioreactor may be provided having a loadable mesh plate holder 200 adapted to receive a plurality of mesh plates 300. In the bioreactor, cells may reside on the mesh plate 300 and grow on the mesh plate 300. The array of mesh plates 300 may be referred to as a culture structure.

Loadable mesh holder 200 is illustrated in fig. 1-4C. Loadable mesh panel holder 200 can accommodate a desired number of mesh panels 300 in a desired spaced orientation. The loadable mesh panel holder 200 illustrated in fig. 1-4 contains 15 mesh panels 300. This is believed to be a suitable number of mesh panels for bioreactor operation.

In general, the loadable mesh holder 200 may enclose an interior space and may be any of a variety of shapes, such as circular, rectangular, and the like. As shown, the loadable mesh holder 200 (on both its interior and exterior) has the general shape of a rectangle with rounded corners. The net panel 300 accommodated by the loadable net panel holder 200 shown in the drawings is rectangular. For ease of description, loadable mesh supports 200 are described herein using orientation indicators such as front and back, side, and horizontal and vertical, which generally correspond to their illustrated orientation or positioning in the assembled bioreactor. However, it will be appreciated that these orientation indications are somewhat arbitrary.

When a screen panel 300 is in place in a loadable screen support 200, there may be a desired amount of space in the vertical direction between the screen panel 300 and its nearest neighbor. In the bioreactor shown, the flow of liquid culture medium can flow through the open spaces in the well plate 300 substantially perpendicular to the flat surface of the well plate 300.

The cell culture area may comprise, for example, about 15 such well plates 300 spaced apart from each other by a sufficient distance such that the well plates do not touch each other (due to possible deflection or bending of the well plates or any other reason) and, if desired, some lateral flow of liquid medium (sideways flow) is possible in the space between adjacent well plates 300.

The loadable mesh holder 200 may be a hollow simple shape, some of which form a complete perimeter (perimeter), such as a circular or rectangular shape (possibly with rounded corners). In general, it is possible that the loadable mesh plate holder 200 may be made in one piece, for example by an additive manufacturing process. However, perhaps more typically, the loadable mesh panel holder 200 may be made of two components that are joined together, such as two molded plastic components. The loadable mesh-plate holder 200 is shown made of two parts that are joined to each other. The loadable mesh panel holder 200 may include a three-sided member (three-sided piece)220 and a closure member 240 that may be combined with the three-sided member 220. The three-sided member 220 may include, in order, a first face section 222A, a rear section 222B, and a second face section 222C opposite the first face section 222A. The closure 240 may include at least one section, which may be considered a front section 242B. As shown, the closure 240 may additionally include a first side section 242A and a second side section 242C. Thus, there is, in order, a first side section 242A, a front section 242B and a second side section 242C. First side segment 242A may be joined with first face segment 222A, and second side segment 242C may be joined with second face segment 222C. However, the closure 240 may also include only the front section 242B. As shown, the front and

rear sections

242B, 222B are substantially parallel to each other, and the first and

second face sections

222A, 222C are substantially parallel to each other, and the first and

second side sections

242A, 242C are substantially parallel to each other. However, other spatial relationships are possible.

The engagement feature may include a deformable tab (deformable tab)250 and may also include an opening 224A in the first face section 222A and a similar opening 224C in the second face section 222C, wherein the openings 224A and 224C are suitably designed to engage the tab 250. The tabs 250 are resiliently deformable between the engaged position shown and a released position in which the deformable tabs 250 flex sufficiently inward to cause disengagement. The tab 250 may include a living hinge and it is further possible that the resiliency elsewhere in the closure 240 may also facilitate the change in size or shape to allow or assist in effecting the disengagement.

The loadable mesh plate holder 200 may include mechanical interface features (illustrated as holes 260) that may be located near the top of the loadable mesh plate holder 200 in the orientation shown. Such apertures 260 may be adapted to mate with handles as described elsewhere herein.

The loadable mesh panel holder 200 may have an upper edge and a lower edge, which may be flat and may be parallel to each other.

Slots and grooves in loadable mesh plate holders, and rounded edges near the slots and grooves

The loadable mesh panel holder 200 may include various grooves and slots that define the position of the mesh panel 300 and provide mechanical support for the mesh panel 300. As shown, the loadable mesh panel holder 200 is capable of accommodating 15 mesh panels 300, but of course other numbers of mesh panels 300 are possible. The separation distance between mesh plates 300 may be selected from a combination of considerations such as the pattern of fluid flow between mesh plates 300, and the desired overall cell packing density in the cell culture region of the bioreactor.

If the loadable mesh panel holder 200 is made in one piece (similar to that shown, but with the three-sided member 220 and closure 240 connected together), it would be possible to insert or remove mesh panel 200 from the loadable mesh panel holder 200. This can be done from the lateral direction.

In the loadable mesh panel holder 200 as described and illustrated above, it is possible to insert or remove the mesh panel 300 from the loadable mesh panel holder 200 when the three-face member 220 and the closure member 240 have been assembled to each other, without the need to disassemble these components from each other. This can be done from the lateral direction. As shown, the first face section 222A has a groove 270 and the second face section 222C has a groove 270. The rear section 222B has a groove 270. The front section 242B has slots through which the mesh panel 300 can pass without disassembling the loadable mesh panel holder 200. The grooves 270 may be substantially coplanar with one another and may have similar or identical dimensions to one another in the vertical direction. The slots 280 may be parallel or coplanar with at least some of the grooves 270. The vertical dimension (vertical dimension) and height (elevation) of the slit 280 may be the same as the vertical dimension and height of the groove 270, but it is sufficient if the vertical dimension and height are only substantially similar to each other. These various features may be combined and interact such that when assembling the loadable mesh panel holder 200, it is possible that the mesh panel 300 is slid into place through the slot 280 and supported by the groove 270 and the slot 280. The desired spacing or distance between the mesh panels 300 is maintained by the material of the loadable mesh panel holder 200 near the groove 270 and the material (engagements) of the loadable mesh panel holder 200 present between the slots 280.

As shown, the closure member 240 has three sides, with two of the

sides

242A, 242C being shorter. One sidewall of the loadable mesh panel holder 200 includes a first face section 222A and a first side section 242A, and similarly, the other sidewall includes a second face section 222C and a second side section 242C. As shown, even the tabs 250 contain a groove that is continuous with the groove 270 in the respective adjacent side segment. However, it will be appreciated that various other designs are possible.

As shown, the grooves 270 in the first face section 222A and the first side section 242A are substantially sharp-edged. The same is true of the grooves 270 in the second face section 222C and the second side section 242C. However, it will be appreciated that other geometries are possible.

At the front of the loadable mesh panel holder 200, a divider or connector of the remaining material may be such that it has a rounded edge generally facing the exterior of the loadable mesh panel holder 200. This helps guide the web plate 300 into the slot 280 during initial insertion of the web plate 300 into the slot 280. The rounded edges may be semi-cylindrical (hemicylindrical). Such rounded edges also help guide the mesh panel 300 into the slot 280 if it happens that any of the mesh panels 300 are not completely flat, e.g., slightly curved out of plane.

At the rear of the loadable mesh plate holder 200, the separation between the individual mesh plates 300 may be such that there are rounded edges generally facing the interior of the loadable mesh plate holder 200. This helps guide the mesh panel 300 to a desired position when inserting the mesh panel 300 into the loadable mesh panel holder 200, for example, near the end of the insertion process. The rounded edges may be semi-cylindrical. The rounded edges may be semi-cylindrical. Such rounded edges also help guide the net panels 300 into the grooves 270 if it happens that any of the net panels 300 are not completely flat, e.g. slightly curved out of plane.

The partition at the front of the loadable mesh plate holder 200 may define a slit through which the mesh plate 300 may pass. The divider at the rear of the loadable mesh plate holder 200 may define a groove into which the mesh plate 300 may enter, but through which the mesh plate 300 may not pass.

With both the rounded edges of the slit 280 and the rounded edges of the groove 270, the semi-cylindrical curvature is only one of various possible curvatures. Fillets (filets) with some other desired radius are also possible. The radius may be smaller or larger than the radius of the semi-cylindrical body. Other shapes of curvature are also possible. Variations in curvature along the length of the slot 280 or groove 270 are also possible.

Screen design

In an embodiment of the invention, referring now to fig. 5A-5H, a bioreactor may have an array of mesh panels 300 within a culture area that serve as tissue scaffolds on which cells (e.g., anchorage-dependent cells) grow. An individual web 300 may itself comprise a plurality of layers of fibers 500, wherein the fibers 500 have an orientation perpendicular to the direction of the fibers in adjacent layers. In particular embodiments of the invention, the number of layers of such fibers 500 in a single web 300 may be, for example, four or five or six layers.

Typical dimensional parameters for such a web 300 may be a fiber diameter of 150 microns and a fiber spacing of 200 microns (as defined in FIG. 5C), the dimension being the distance from the edge of one fiber to the nearest edge of the nearest fiber in the same plane. The fibers 500 in one layer may be staggered relative to the fibers 500 parallel to them and in a different layer of the web 300, or alternatively, not necessarily. The fibers 500 may be spaced from each other by a suitable distance such that during initial cell seeding, cells are deposited on the fibers 500 and do not contact cells on adjacent fibers 500. However, the spacing of the fibers 500 may be such that after a certain number of cells proliferate, cells growing on nearby fibers 500 may contact each other or grow relative to each other (a condition known as confluency).

More specifically, the fibers 500 may be present in two mutually perpendicular directions, and may be staggered in both mutually perpendicular directions. This is further illustrated in fig. 5D. Each web sheet may include a plurality of fibers forming a first layer having parallel fibers oriented in a first direction, and may include a second layer having a plurality of parallel fibers oriented in a second direction, the second direction being perpendicular to the first direction, the fibers in the second layer being one layer attached to the fibers in the first layer. At least some of the webs include a plurality of fibers, and within a single web, the fibers are arranged in sequence in at least first, second, third, and fourth layers, the fibers within each layer being substantially parallel to each other, wherein the fibers in the first layer are substantially parallel to the fibers in the third layer, the fibers in the second layer are substantially parallel to the fibers in the fourth layer, and wherein the third layer is positioned out of alignment with the fibers of the first layer and the fourth layer is positioned out of alignment with the fibers of the second layer when viewed perpendicular to the planar surface of the web. More specifically, with respect to this viewing direction, the fibers may be located midway between their non-aligned parallel fibers. Such a configuration may help prevent cells from falling off the mesh plate, particularly during initial seeding, while still providing space for liquid media to occupy and flow through, and space for cells as they proliferate.

The well plate 300 may have a certain number of layers, for example four layers, and a culture medium for contacting cells may be used on both surfaces of the four-layer configuration, and a culture medium may also be used in the openings existing between the layers. It is believed that all of the features of this situation more closely resemble the phenomena occurring in natural tissue, which may be referred to as a three-dimensional environment. It is believed that the three-dimensional aspect of embodiments of the present invention is more favorable to the proliferation and expansion of cells than a two-dimensional environment. However, it is not intended to be limited to this explanation.

Referring now to fig. 5E-5H, plan views (plan views) of the screen of fig. 5A-5D are shown, wherein different numbers of layers are shown. (for clarity of illustration, only a simple representation of less than all of the fiber layers is shown.) in FIG. 5E, only one layer is shown. FIG. 5F shows the first and second layers with the second layer having a fiber orientation perpendicular to the fiber orientation of the layer shown in FIG. 5E. Fig. 5G adds a third layer with fiber orientation parallel to that of the first layer and with fiber position staggered relative to the first layer. It can be observed that there is not much perspective (see through) and that the width of the perspective area is smaller than the fiber diameter. Finally, FIG. 5H shows four layers, with a newly shown fourth layer having fibers that are parallel to and staggered relative to the fibers of the second layer. It can be observed that the size (size) of the see-through area is even smaller than the area visible in fig. 5G, and the size (dimension) of the see-through area is smaller than the fiber diameter. It is believed that the small size of the see-through area relative to the fiber diameter helps retain liquid that may be deposited on the mesh sheet during sowing.

In another embodiment of the invention, a mesh sheet 300 comprising six layers of filaments (filaments) may be provided. As in the four-ply net shown, adjacent plies may have fiber orientations that are perpendicular to each other. However, for an offset between layers having the same fibre direction, the use of six layers will provide more possible options. For example, the offset distance between one layer and another layer having the same fiber orientation may be one third of the interfiber spacing, and the offset between one layer and another layer having the same fiber orientation may be one third of the interfiber spacing in the opposite direction. For example, using the same fiber diameters and fiber spacings as already shown and the offsets just described, it is possible to build a six-ply web in which there is no line-of-sight path (see-through) in the direction perpendicular to the entire surface of the web.

Mesh panel 300 may be formed by the programmed deposition of heated filaments of polymer, similar to that described in commonly owned U.S. patent 8,463,418. The polymer may be a suitable biocompatible polymer, such as polystyrene. The mesh sheet 300 is non-woven. Alternatively, mesh panel 300 may be woven, if desired. The overall shape of the mesh panel 300 may be flat and rectangular.

Referring now to fig. 5I, it is further possible that the mesh panel 300 has a non-uniform thickness. For example, the thickness of the mesh panel 300 near the edge of the mesh panel 300 sliding in the groove may be relatively thin, and the thickness of the mesh panel 300 away from the edge of the mesh panel 300 may be relatively thick. The thicker central portion may provide more space to accommodate more cells during culture than in the case of a mesh plate 300 of uniform thickness.

In some prior art cell culture techniques, such as Petri dishes (Petri dish), a layer of cells is grown on a flat surface and subjected to a substantially two-dimensional environment. Even if in this case there is sufficient supply of nutrients and waste removal, this two-dimensional environment is inherently different from the environment in which cells naturally grow, i.e., the three-dimensional environment.

In an embodiment of the invention, the cells are seeded by attaching to individual fibers of a mesh sheet. The fiber-to-fiber size of the mesh sheet may be large enough such that when the isolated cells are initially attached to the fibers, at least some of the cells do not normally contact other cells. After the cells have propagated, and possibly several layers of cells are produced at the fiber where only one layer of cells was originally attached, the outermost layer of cells may remain independent of the next fiber, or some new cells may contact other cells attached to other fibers, i.e. some fiber bridging (called confluency) may occur. In embodiments of the invention, the web sheet may have a specific number of fibrous layers, for example four layers. Media may be used on both surfaces of the four-layer construct for contacting cells. In any event, it is believed that all of the features of this situation more closely resemble the phenomena occurring in natural tissue, which may be referred to as a three-dimensional volumetric environment. It is believed that the three-dimensional aspect of embodiments of the present invention is more favorable to the proliferation and expansion of cells than a two-dimensional environment. However, it is not intended to be limited to this explanation.

The cell culture zone may comprise, for example, about 10 to 15 such mesh plates spaced apart from each other by a sufficient distance such that the mesh plates do not contact each other and liquid can flow between the mesh plates.

In terms of biological parameters, a well plate for use in an embodiment of the invention may have an area (length dimension x width dimension) of about 7000mm 2, and the area of the portion of the well plate exposed to perfusion (excluding edges in grooves or slits) may be about 6300mm 2. The web may be formed from four layers of fibers, with the layers alternating in the direction as described elsewhere herein. Alternatively, five or six layers of fibers or some other number of layers of fibers may be used. On such a well plate, an initial seeding of about 800,000 cells can be deposited, and thus, if 12 well plates are used, the total number of seeded cells is 960 ten thousand cells. At the end of the culture, the cell population can expand approximately 25-fold.

In order to culture and seed cells onto the scaffold, it is considered desirable that the surface properties of the scaffold material be hydrophilic. Some polymers are useful scaffold materials and are inherently less hydrophilic than desired. In general, it may be desirable for a drop of water or aqueous liquid to diffuse on and within the screen after deposition onto the screen. In embodiments of the invention, after the web is formed by appropriately positioning the fibers, the polymeric scaffold material, such as polystyrene, may be plasma treated. Plasma processing may be accomplished in a Plasma chamber apparatus, such as that available from Harrick Plasma (Harrick Plasma), inc. In such an apparatus, a gas at sub-atmospheric pressure (low sub-atmospheric pressure) is subjected to radio frequency electromagnetic field radiation to generate a plasma within the process chamber at near ambient temperature. The gas used may be argon, oxygen, air, hydrogen, nitrogen, mixtures of these gases or other options, or no special gas components need be added at all. Such treatment may clean the surface. Such treatments may effect surface modification, which may include changing the surface energy of the surface in either direction depending on the details of the treatment. Surface modification may involve attaching atoms or functional groups (e.g., oxygen-containing functional groups) to the polymer. Surface treatment may cause the polymer surface to become more hydrophilic than before the treatment. For example, the surface properties of the treated polymer surface may be such that the contact angle of the treated polymer with pure water is less than 40 degrees or less than 50 degrees. (a smaller contact angle indicates a more hydrophilic state and a larger contact angle indicates a more hydrophobic state.) after the plasma treatment, it may also be suitable to perform the electrostatic charge removal process at the end of the plasma treatment. Such a treatment may remove or neutralize static charges that may remain at the end of the plasma treatment. This treatment can be performed using a static eliminator from Keyence Corporation (itasca, illinois). Such devices discharge a stream containing both positively and negatively charged ions to the workpiece, and any ions needed to neutralize the electrostatic charge contact the surface and neutralize the electrostatic charge.

Pusher attachment and slit for positioning net plate

Referring now to fig. 7A-7C, as described herein, in an embodiment of the present invention, the loadable mesh panel holder 200 may include a positioning slit 390 that may be used to push the mesh panel 300 into a desired position or to define the position of the mesh panel 300. Such positioning slits 390 may be generally perpendicular with respect to the general direction of the loadable mesh plate holder 200 and the bioreactor. The positioning slot 390 may intersect other slots and grooves provided in the loadable mesh panel holder 200.

The pusher attachment 400 may also be provided as an attachment (for use before or after the actual culture). To push the screen plate 300 into the loadable screen plate holder 200 until it contacts a stop such as the base of the groove 270, it is possible to use a pusher attachment 400 to one side of the loadable screen plate holder 200. To push the mesh plate 300 out of the loadable mesh plate holder 200, it is also possible to use the pusher attachment 400 on the opposite side of the loadable mesh plate holder 200, for example when a culture is completed or it is desired to remove the mesh plate 300. The positioning slots 390 on one side of the loadable mesh panel holder 200 may have the same or similar size and spacing as the positioning slots 390 on the other side of the loadable mesh panel holder 200, which would enable a single pusher attachment 400 to be pushed in both directions.

To facilitate the described pushing, the pusher attachment 400 may have some dimensional relationship with appropriate features of the loadable mesh deck holder 200. The bosses 420 on the pusher appendage 400 may be sized and may be appropriately spaced relative to each other so that they may fit into the positioning slots 390 of the loadable mesh plate holder 200. The height of the bosses 420 on the pusher attachment 400 may be large enough so that the net panel 300 may be pushed to a desired extent.

Storage screen holder

Referring now to fig. 8A and 8B, in addition to providing a loadable mesh holder 200, a storage mesh holder 900 may also be provided. The storage screen holder 900 may have some geometric similarities to the loadable screen holder 200, but may also differ in certain aspects.

The storage screen holder 900 may have dimensions and features suitable for receiving the screens 300 in a geometric shape such that the screens 300 in the storage screen holder 900 are coplanar with their final respective positions in the loadable screen holder 200. In particular, the storage screen holder 900 may have an interior recess 906 adapted to receive the screen 300. The size and location of such grooves may be similar or identical to corresponding features in the loadable mesh holder 200. As shown, the grooves 906 on the sides of the storage screen holder 900 have rounded inner edges, through which grooves 906 the screen can be loaded into the storage screen holder 900, which can help the screen 300 enter those grooves.

The storage screen holder 900 may have an opening 910, the opening 910 being adapted to allow the pusher 1100 to occupy space and contact a trailing edge (trailing edge) of the screen 300, if necessary, and to push the screen 300 out of the storage screen holder 900 into the loadable screen holder 200. The size and location of the opening 910 may be such that the pusher 1100 never actually contacts any boundary of the opening 910 when the pusher 1100 is in any of its allowable positions.

The side of the storage screen holder 900 intended to abut the loadable screen holder 200 is shown without a stop. This may allow the screen panel 300 to more easily slide out of the storage screen holder 900 and load the loadable screen panel holder 200. This empty space allows the pusher 1100 to push the mesh panel 300 into the loadable mesh panel holder 200 until the pusher 1100 approaches or contacts the corresponding surface of the recess 210. This helps to achieve the desired positioning of the mesh panel 300 in the loadable mesh panel holder 200.

As shown, storage screen holder 900 has an upper surface 920 that may facilitate structural connectivity of storage screen holder 900. Of course, variations of these design features are possible.

Fig. 8B shows several screens 300 in place in a storage screen holder 900. As shown, the dimensional relationship between the width of the mesh panel 300 and the width of the storage mesh panel holder 900 is such that the mesh panel 300 extends beyond the storage mesh panel holder 900 to the extent that the mesh panel 300 will occupy a small amount of space in the loadable mesh panel holder 200 when the loadable mesh panel holder 200 and the storage mesh panel holder 900 are in their operative relative configuration. As a result, when the pusher 1100 tries to advance the mesh panel 300, the leading edge of this mesh panel 300 will have passed the interface between the storage mesh panel holder 900 and the loadable mesh panel holder 200 and this will eliminate any possibility that the mesh panel 300 gets mechanically stuck on the interface between the storage mesh panel holder 900 and the loadable mesh panel holder 200.

Assembly of a storage screen holder and a loadable screen holder

Referring now to fig. 9A, 9B, 10A and 10B, a storage screen holder 900 and a loadable screen holder 200 are shown. Fig. 9A, 9B show the storage net holder 900 and the loadable net plate holder 200 opposite to each other when assembled for use. FIGS. 10A, 10B show an exploded storage screen holder 900 and a loadable screen holder 200 separated from each other.

The storage screen holder 900 may have suitable dimensions and features such that the storage screen holder 900 may be aligned with a loadable screen holder 200 to achieve the following relationship: the panels 300 in the storage panel holder 900 are coplanar with their respective positions in the loadable panel holder 200. For example, some of the outer dimensions of the storage screen holder 900 and the loadable screen holder 200 may be identical to each other to facilitate such alignment, and the inner dimensions (e.g. dimensions and positions of grooves and slots) with respect to the vertical positioning of the screen 300 may be identical when comparing the loadable screen holder 200 and the storage screen holder 900. It would also be possible to use alignment pins or connectors that directly connect the storage screen holder 900 and the loadable screen holder 200 to each other, or similar features known in the art, although not shown here. Alignment using a seed tray would also be possible as described herein. The dimensions and other relationships between the mesh panel 300 and the grooves in the storage mesh panel holder 900 and the loadable mesh panel holder 200 may be such that the mounting of the mesh panel in the groove is loose enough that the mesh panel 300 may easily slide between the storage mesh panel holder 900 and the loadable mesh panel holder 200 without applying a substantial amount of force.

As shown, storage screen holder 900 has an opening 910 on one side for pusher 1100 to pass through.

The interval between the grooves in the storage screen holder 900 and the loadable screen holder 200 in the vertical direction and the vertical positions of the grooves may be the same as each other so that the screen 300 may be easily slide-loaded from the grooves in the storage screen holder 900 into the grooves of the loadable screen holder 200.

FIGS. 10A (full view) and 10B (cross-sectional view) show a storage screen holder 900 and a loadable screen holder 200 similar to FIGS. 9A and 9B, except that for clarity of illustration the storage screen holder 900 and the loadable screen holder 200 are separated from each other.

Seeding tray

Referring now to fig. 11A and 11B, as shown, both the lower portion of the storage screen holder 900 and the lower portion of the loadable screen holder 200 may be contained in a close-fitting sowing tray 1300. The sides of the seeding tray 1300 may abut respective sides of the lower portions of the storage screen holders 900 and the loadable screen holders 200, thereby providing geometric alignment of those screen holders with respect to each other. Such mounting and relationship may also provide for alignment of the positions of the storage screen holder 900 and the loadable screen holder 200 relative to each other and relative to other components of the machine.

The seeding tray 1300 may have open spaces 1310 on its sides that are adapted to accommodate all desired positions of the pusher 1100 to allow the pusher 1100 to pass through the sides of the seeding tray 1300 at all allowable positions of the pusher 1100. Open space may also be provided for any components located near the pusher 1100 or attached to the pusher 1100. In other designs, the bottom of the seeding tray 1300 may have suitable openings to accommodate all desired positions of the pusher 1100.

The sowing tray 1300 may have a grip (griping handle)1320 protruding therefrom. The grip 1320 may allow a user to grasp the grip 1320 to carry the seeding tray 1300 and the loadable mesh panel holder 200 and the storage mesh panel holder 900 and any mesh panels 300 contained therein while the user's hand is kept at a distance from those components. This can help maintain the sterility of the components during handling of the components after they are removed from their sterile packaging.

The sowing tray 1300 may be configured and dimensioned such that the sowing tray 1300 is aligned with the storage screen holder 900 and the loadable screen plate holder 200 in a desired direction, but allows the loaded and sowed loadable screen plate holder 200 to be lifted vertically even when the storage screen holder 900 and the loadable screen plate holder 200 are in the sowing tray 1300. For example, the sowing tray 1300 may be such that the bottom surfaces of the storage screen holder 900 and the loadable screen holder 200 are coplanar with each other, and one side of the storage screen holder 900 is in contact with a corresponding side of the loadable screen holder 200, and one side of the storage screen holder 900 is coplanar with one side of the loadable screen holder 200, and the opposite side of the storage screen holder 900 is coplanar with the corresponding side of the loadable screen holder 200.

It is possible that the loadable mesh plate holder 200 and the storage mesh plate holder 900 can slide vertically relative to each other, in a sense that if the loadable mesh plate holder 200 and the storage mesh plate holder 900 are already present in the sowing tray 1300, it is possible to slide the loadable mesh plate holder 200 into or out of position in the sowing tray 1300 using a substantially vertical movement. This is possible if the seeding tray 1300 already provides the required alignment and registration between the loadable mesh plate holder 200 and the storage mesh plate holder 900. Thus, there may not be a direct bond (engagement) between loadable mesh holder 200 and storage mesh holder 900, which would prevent relative vertical movement between these two components, and when mesh panel 300 is fully in loadable mesh holder 200, no mesh panel would extend through the interface between loadable mesh holder 200 and storage mesh holder 900 to impede relative vertical movement between these two components.

In embodiments of the invention, there may be some geometric relationship between the loadable mesh panel holder 200 and the storage mesh panel holder 900 and mesh panel 300. The screen plate 300 may have a screen plate width WS in a moving direction to follow the screen plate 300 when sliding from the storage screen holder 900 to the loadable screen holder 200. The storage screen holder 900 may have an outer dimension, SSW, in the same direction. The loadable mesh plate holder 200 may have the outer dimension of the LSW in the same direction. SSW < WS < LSW is possible. This means that the net panel 300 may be able to fit completely within the loadable net panel holder 200. This allows the loadable mesh plate holder 200 to be removed from the sowing tray 1300 with a simple vertical movement after all the mesh plates 300 are slid into the sowing tray 1300, regardless of whether the storage mesh plate holders 900 are moved simultaneously or not. Moreover, this relationship provides that prior to a seeding operation, when all of the mesh panels 300 are in the storage mesh panel holder 900, the mesh panels 300 have already extended slightly into the wall thickness of the loadable mesh panel holder 200. This extension provides a starting point where the mesh panel 300 has extended across the interface and across a groove in the loadable mesh panel holder 200, which makes it less likely that mesh panel 300 will become stuck at the interface between storage mesh panel holder 900 and loadable mesh panel holder 200. It is possible that when the mesh plate 300 is in the storage mesh plate holder 900, the mesh plate 300 only extends into the wall thickness of the loadable mesh plate holder 200, but not beyond the wall thickness, in order to avoid hindering the movement of the pipette 2020 and dispensing of fluid from the pipette 2020 into the inner space of the loadable mesh plate holder 200.

This case can also be described as the case where one of the mesh panels 300 may reside in a loadable mesh panel holder 200 without extending outside the outer envelope of the loadable mesh panel holder 200, but when said mesh panel 300 resides in a storage mesh panel holder 900, said mesh panel 300 extends outside the outer envelope of the storage mesh panel holder 900.

This relationship can be further described as a relationship such that one of the mesh panels 300 may reside in a loadable mesh panel holder 200 without extending outside the outer envelope of the loadable mesh panel holder 200, but when said mesh panel 300 is located in a storage mesh panel holder 900, said mesh panel 300 may extend outside the outer envelope of the storage mesh panel holder 900. If the loadable screen holder 200 or storage screen holder 900 has a substantially flat outer surface in addition to the locally inwardly directed feature, the envelope may be considered to be a plane that contacts the adjacent outer flat portion.

Auxiliary components related to sterility

In an embodiment of the present invention, a storage screen holder 900 and a loadable screen holder 200 assembled to each other, and a screen 300 present in the storage screen holder 900 may be provided, wherein all of these components are sterile and packaged in sterile packaging prior to performing a seeding procedure. All of these components may be assembled with each other under sterile conditions and may be packaged in sterile packaging and shipped as is. Transportation presents the possibility that the assembly may occupy any orientation of space during transportation. It is desirable that in this case, the mesh plates 300 are restrained so that they cannot slide or change their positions. Thus, referring now to fig. 12A, a stop (stop)1400 may be provided in such an assembly, the stop 1400 being adapted to prevent the screen 300 from being removed from the storage screen holder 900, for example, during transport of such an assembly. The stop 1400 may be a flexible or partially flexible sheet that contacts a portion of the mesh panel 300 and a portion of the interior of the loadable mesh panel holder 200 in a manner that the stop 200 prevents movement of the mesh panel 300 when the stop 1400 is in place. The stopper 1400 may have elasticity and a suitable shape to keep itself in a position that may prevent movement of the mesh plate 300, and the stopper 1400 may be easily removed by a user prior to the cell seeding process, i.e. prior to sliding any mesh plate 300 from the storage mesh plate holder 900 to the loadable mesh plate holder 200. The stop 1400 may be a piece of cardboard or other paper-made material or polymeric material (e.g., polystyrene) that is folded into an angled shape as shown in fig. 12A. As shown, the stop 1400 may be V-shaped with a fold or bend. It may be a truncated V-shape with two folds or bends. It would also be possible for the stop 1400 to be resiliently bent or bent into a more general shape. The stopper 1400 may be sterile and may be sterilized at the same time as the other components of the package.

It is also possible to provide a lifting handle (lifting handle) adapted to engage with and lift the loadable mesh panel holder 200. An example of a handle 1500 is shown in isolation in fig. 12B, and in conjunction with the loadable mesh panel holder 200 in fig. 12C. The handle 1500 may be adapted to engage the aperture 260 in the loadable mesh panel holder 200 and disengage when desired. The handle 1500 may be deformable between two configurations (configurations) such that in a first configuration it can be moved into a space adjacent the loadable mesh panel holder 200 or inside the loadable mesh panel holder 200, and in a second configuration it may be engaged with the loadable mesh panel holder 200. Handle 1500 may be supplied under sterile conditions. The handle 1500 may have a geometry such that the hand of the user grasping the handle is kept a distance from the actual loadable mesh panel holder 200 and mesh panel 300 contained therein, thereby helping to maintain the sterility of the loadable mesh panel holder 200 and mesh panel 300. The handle 1500 may be sterile and may be packaged with other components or separately.

Pusher and motion control system

A pusher 1100 may be provided, the pusher 1100 being adapted to push the mesh panel 300 from a storage position in the storage mesh panel holder 900 to a loading position in the loadable mesh panel holder 200. Such a pusher is shown in fig. 13A and 13B. The pusher 1100 may be attached and detached from the motion control mechanism that drives and positions it. The pusher 1100 may be a single-use component that may be provided in a packaged form suitable for maintaining its sterility in a sterile state. Pusher 1100 may be provided in a sterile state to help avoid contamination of web 300, and may be shipped in a suitable sterile package.

As shown, the pusher 1100 may enter the storage screen holder 900 through one side of the storage screen holder 900. The storage screen holder 900 may have suitable opening or openings to allow the pusher 1100 to pass through the side of the storage screen holder 900 in all allowed positions of the pusher 1100 so that the pusher 1100 can reach the screen 300 as needed to touch and move the screen 300. The seeding tray 1300 may also have suitable openings 1310.

In fig. 13A to 13B, the components are exploded for convenience of illustration. It is shown that the pusher 1100 may be attached to a motion control mechanism at a defined location and retained by magnetic force. One or the other of the pusher 1100 and the corresponding motion control mechanism may comprise magnetic components, and the other may comprise suitable magnetic or metallic components. The interface may include features or dimension controlled surfaces to define the position and orientation of the pusher 1100 when it is connected to the motion control system. Such features may be, for example, posts (posts) and corresponding recesses, or may be flat, dimensionally controlled surfaces, or may be other types of dimensionally controlled surfaces. The magnetic feature may be part of the dimensionally controlled surface or may be separate from the dimensionally controlled surface.

As shown in fig. 13A to 13B, the pusher 1100 may be a member that contacts and pushes the mesh plate 300, and may be horizontally oriented. A support 1110 may further be provided which may be connected to or part of the motion control system. As shown, the pusher 1100 may have two locating pins 1120 that may mate with corresponding locating holes or recesses 1130 in the support 1110 to define the position of the pusher 1100. The magnet 1140 may be permanently mounted in the support 1110. Removable posts 1150 may further be provided, some of which may occupy or pass through holes 1160 in the pusher 1100. The post 1150 may be made of a material that is attracted by the magnet 1140. The interaction of the pusher 1110 with the associated components, including magnetically engaged components, may be such that, if a force or deflection is accidentally applied or imposed on the pusher 1100, the components of the magnetic joint may partially tilt or separate to absorb the force or deflection and may return to their original position upon removal of the force or deflection caused by magnetic attraction. The pusher 1100 may be provided in a sterile state and may be a single-use component.

The pusher 1100 may be mounted on a motion control system adapted to allow or direct movement of the pusher 1100. For example, the pusher 1100 may have freedom of movement in a desired direction to push the mesh panel 300 from the storage mesh panel holder 900 into the loadable mesh panel holder 200. Such a degree of freedom may be the only degree of freedom of movement provided for the pusher 1100. Alternatively, other or additional degrees of freedom of movement may be provided. The pusher 1100 may be driven in the appropriate direction of motion by a drive system or motion control system 2060. The movement of the pusher 1100 may be in a left-right direction, as viewed from the front of the system.

The pusher 1100 may be substantially horizontal in its orientation during use, and its movement may be horizontal, and its length may be at least the size of the mesh panel 300 in the direction along the movement direction of the mesh panel during sliding. When the mesh plate 300 is in place in the storage mesh plate holder 900 or the loadable mesh plate holder 200, the pusher thickness (in the vertical direction) of the pusher 1100 may be less than twice the mesh plate-to-mesh plate spacing distance of the mesh plate 300. However, other geometries and orientations of the pusher 1100 are possible.

Referring now to fig. 14A-15, a motion system may be provided that is adapted to provide a desired relative position of the pipette 2020 and pusher 1100, as well as the loadable mesh plate holder 200 and mesh plate 300. During loading of a set of well plates 300 into a loadable well plate holder 200, a plurality of well plates 300 may be present in the loadable well plate holder 200, and at a given time, deposition of cells onto any uppermost well plate 300 in well plates 300 in the loadable well plate holder 200 may be accomplished by pipette 2020.

In an embodiment of the invention, the motion control system may be such that one axis of motion is provided by the means for moving the assembly of the screen 300 and the

screen holder

200, 900, another axis of motion is provided by the means for moving the pusher 1100, and yet another axis of motion is provided by the means for moving the pipette 2020. The three axes of motion may be perpendicular to each other. For example, as shown, the drive moving the assembly of the screen holder and the screen can move them in a vertical direction. The driver moving the pusher 1100 may move the pusher 1100 in a horizontal direction, which is a left-right direction in the perspective view of fig. 15. The drive moving the dispenser or pipette 2020 may move them in a horizontal direction, front to back in the perspective view of fig. 15. As shown in fig. 15, the pusher 1100 can be driven in the horizontal direction (left-right direction in the illustrated view) by the horizontal driver 2060. There may be a platform that houses the loadable mesh plate holder 200 as well as the storage mesh plate holder 900 and the reservoir 1000, and that can be driven in a vertical direction by a vertical drive 2070. The dispenser 2000 may be driven in a horizontal direction (back and forth in the illustrated view) by a horizontal driver 2080.

In the system shown in fig. 14A to 15, the vertical height (vertical elevation) of the loadable mesh plate holder 200 during cell deposition need not be the same as the vertical height during sliding of mesh plate 300 from storage mesh plate holder 900 to loadable mesh plate holder 200. As shown, the pusher 1100 has a fixed height in the vertical direction while being able to translate appropriately in the horizontal direction to cause the desired sliding of the otter board 300 (due to its horizontal drive 2060). The motion control system may be suitably operated to suitably position the seeding tray 1300 and the storage screen holder 900 and loadable screen holder 200 to cause the desired sliding of the screen 300, and may also be suitably operated to seed the cells such that the exposed screen 300 in the loadable screen holder 200 is positioned at a desired position or height relative to the pipette 2020. Therefore, in the illustrated system, the pusher 1100 does not have to have a vertical position adjustment mechanism, and the pipette 2020 does not have to have a vertical position adjustment mechanism. As shown, while it is sufficient to provide only one vertical movement mechanism, it is also possible to provide more than one vertical movement mechanism, if desired.

Other arrangements than those described are possible if desired. For example, it would be possible to provide more freedom of movement for the screen holder assembly, and less freedom of movement for the pusher 1100. Alternatively, it would be possible to provide more freedom of movement for the pipette 2020, and less freedom of movement for the loadable mesh plate holder 200, for example.

The apparatus may also include automatic controls, drive systems, motion control systems, and software adapted to operate the described components in a desired sequence. A stepper motor may be used. Sensors such as encoders may be included as desired. Feedback control or servo systems may be provided.

Dispenser

Referring now to fig. 14A-15, a dispenser 2000 adapted to dispense cells for seeding a mesh plate 300 is shown. Dispenser 2000 may be adapted to dispense a fluid, such as a liquid (which may be a culture medium) in which cells are suspended. Such a device may resemble a pipette for dispensing biological samples or substances into a plate, such as a conventional microtiter plate (containing 96 wells, 48 wells, or other number of wells), which is commonly used for various biological assays, e.g., for high throughput screening. Dispenser 2000 may include an array of pipettes 2020 in a uniformly spaced arrangement, or any other desired arrangement. For clarity of illustration in fig. 14A-15, seven pipettes 2020 are shown, but any number of pipettes 2020 may be used. Pipettes 2020 are shown as being identical to one another, but they may be physically different from one another if desired. They may operate the same as each other or they may operate differently from each other if desired. The dispensing action of pipette 2020 may be determined by an automated system, which may be computer controlled. Fluid may be dispensed at any given time onto the uppermost mesh panel 300 in the stack in the loadable mesh panel holder 200. An appropriate amount of fluid may be dispensed containing an appropriate number of cells to accomplish the desired seeding. Dispensing may be performed to avoid causing excessive dripping of fluid from the screen 300. In fig. 14A to 14C, some parts of the entire system are omitted for clarity of illustration. It will be appreciated that although the term "pipette" is used exemplarily, it will also be possible to use other forms of fluid dispensing, including fluid dispensing in connection with inkjet technology.

A fluid reservoir 1000 may also be provided. Reservoir 1000 may contain a sufficient amount of fluid to be deposited on mesh panel 300 as desired during the seeding process. Such fluids may comprise a liquid and cells suspended in the liquid.

The reservoir 1000 may be mounted on the same platform as the storage screen holder 900 and the loadable screen holder 200 such that when the storage screen holder 900 and the loadable screen holder 200 are raised and lowered, the reservoir 1000 is also raised and lowered. If the tip of pipette 2020 is below the surface of the fluid in reservoir 1000, reservoir 1000 may be positioned such that pipette 2020 may withdraw fluid from reservoir 1000.

During operation of the system, it is possible to cause oscillations in the liquid level in pipette 2020 by alternately drawing fluid from reservoir 1000 into pipette 2020, and then ejecting fluid from pipette 2020 into reservoir 1000. This oscillation of the liquid level in pipette 2020 may be used for the purpose of generating motion in the fluid in reservoir 1000. This motion may be used to mix the fluid in reservoir 1000 and counteract any possible tendency of cells suspended in the fluid in reservoir 1000 to settle or stratify in the fluid in reservoir 1000. It is contemplated that after several oscillations of the liquid level in the pipette 2020, the fluid in the reservoir 1000 may have a more uniform concentration of cells than without such mixing, and the fluid that is eventually drawn into the reservoir 2020 to be dispensed onto the well plate 300 may be more representative of the desired concentration of cells than without such mixing.

For example, during mixing, the flow into or out of the pipette may be greater (perhaps several times greater, or even an order of magnitude greater) than the flow during dispensing of the droplets onto the screen 300. During this oscillatory flow, the flow into pipette 2020 may be different than the flow out of pipette 2020.

As shown, seven pipettors 2020 are shown, dispensing fluid onto well plate 300,

and the pipettes 2020 are arranged in a single row. However, it will be appreciated that other numbers and arrangements of pipettes thereof are possible. Pipettes 2020 may or may not be physically identical to one another.

Pipettes 2020 may operate the same or different from each other.

Sowing method

Embodiments of the invention may include a method of seeding a cell onto a plurality of well plates in a well plate holder.

The placement of cells on the screen 300 can be controlled by instructions given to the motion control system and the pipette 2020. The density of the seeded cells may be uniform over the entire area of the mesh plate 300, and all mesh plates 300 may be seeded identically to each other. However, other cell distributions will be possible if desired. For example, the density of deposited cells may be varied depending on the location within the well plate 300, if desired. Also, different distributions of seeded cells can be provided on different mesh panels 300, if desired. Any such changes may be implemented by appropriate software or instructions to the system. Even if the system is operated only to provide evenly distributed seeding, such operation may provide assurance that even seeding is achieved, with a greater degree of assurance being possible than with certain other cell seeding procedures or techniques.

As an example, a procedure for seeding cells onto a stack of mesh plates 300 in a loadable mesh plate holder 200 may comprise the steps of:

load the screen 300 into the storage screen holder 900. This loading may be done manually and all available recesses in the storage screen holder 900 may be filled with the screen 300. At this point in the process, the web 300 may be dry.

Align and abut the storage screen holder 900 and the loadable screen holder 200, aligning the two screen holders or components thereof in position in the seeding tray 1300 or machine.

Place the storage screen holder 900 and the pusher 1100 relative to each other so that the pusher 1100 can push the lowest screen 300 in the storage screen holder 900 without pushing any other screen 300 in the storage screen holder 900.

Sliding the lowest screen 300 from the storage screen holder 900 into the loadable screen holder 200. The pusher 1100 may then be retracted to a retracted position.

Raise the platform containing the reservoir 1000 and storage screen holder 900 and loadable screen holder 200 so that the tip of pipette 2020 is below the liquid level in reservoir 1000. The liquid level in the pipette 2020 is oscillated several times. The platform is lowered so that the pipette 2020 can be moved out of the vicinity of the reservoir 900.

Move the pipette 2020 (forward in the orientation shown), bring the pipette 2020 over the interior of the loadable mesh plate holder 200, and then raise the platform (comprising the loadable mesh plate holder 200 and the storage mesh plate holder 900) until the tip of the pipette 2020 is in the desired position relative to the mesh plate 300, which is currently exposed (uppermost) in the loadable mesh plate holder 200.

Dispense fluid containing liquid and cells from pipette 2020 onto well plate 300, which is currently exposed (uppermost) in loadable well plate holder 200.

Lower the platform, disengage (clear) the tip of pipette 2020 from the wall of the loadable plate holder 200, and move the array of pipettes 2020 back to reservoir 1000. The platform is raised so that the tip of pipette 2020 is below the surface of the fluid in reservoir 1000. The liquid level in the pipette 2020 is oscillated and ends when the pipette 2020 is full.

Repositioning the storage screen holder 900 and the pusher 1100 so that the pusher 1100 contacts the lowest screen 300 remaining in the storage screen holder 900 and pushes the screen 300 into the loadable screen holder 200.

Move the platform vertically and move the array of pipettes 2020 horizontally so that pipettes 2020 are in place to deposit fluid onto the newly positioned screen 300.

Deposit cells onto the newly positioned well plate 300 in the loadable well plate holder 200.

These steps are continued in sequence until all the plates 300 have been transferred from the storage plate holder 900 to the loadable plate holder 200 and have been seeded with cells. In order to perform these steps for a specific screen plate 300, the vertical position of the storage screen plate holder 900 may be appropriately adjusted so that the pusher 1100 pushes the specific screen plate 300. The vertical position of the pusher 1100 with respect to the storage screen holder 900 can be changed for each individual screen 300 to be moved.

As the program progresses, the storage screen holder 900 will start emptying the screen 300 at its bottom until the screen 300 is emptied, and the loadable screen holder 200 will start filling the screen 300 at its bottom until it is full of screens 300.

It will be appreciated that the vertical height of the platform may vary depending on which well plate 300 receives the dispensed fluid from the pipette 2020. Also, the vertical height of the platform when sliding the well plate 300 from the storage well plate holder 900 to the loadable well plate holder 200 need not be the same as the vertical height of the well plate 300 when the pipette 2020 dispenses fluid onto the well plate 300.

It is understood that the liquid level in the reservoir 1000 may change during this process. Accordingly, the vertical height of reservoir 1000 when filling pipette 2020 or when performing fluidic oscillations in pipette 2020 may be suitably selected such that the tip of pipette 2020 is below the level of fluid in reservoir 1000. It is possible to calculate the volume of fluid remaining in the reservoir 1000 from the steps already performed in the process and adjust the process parameters accordingly. Such adjustments may relate to the relative vertical position of the reservoir 1000 and pipette 2020, or the flow rate parameter of the aspiration or ejection of fluid, or any other relevant parameter, during the time that fluid is aspirated into or ejected from the pipette 2020.

Pipette 2020 may not move relative to well plate 300 during the actual time liquid and cells are dispensed to well plate 300; or, alternatively, the moving and dispensing may occur simultaneously, if desired. Alternatively, it would be possible for the pipette 2020 or similar dispenser to move in a certain predetermined path, e.g. a raster path, relative to the screen 300 during deposition. As a further alternative, it would also be possible to deposit a spray of cell-containing liquid from a dispenser which may be stationary or moving relative to the well plate. The amount of fluid dispensed from a single pipette 2020 in a single dispense can be controlled as desired. As shown, where only seven pipettes are dispensed onto the screen 300 with substantial spacing between the pipettes 2020, it is expected that a single dispensed droplet from a single pipette 2020 will land on the screen 300 as a distinct droplet different from the droplets deposited by adjacent pipettes 2020. However, it is also contemplated that droplets deposited on screen 300 will break up and merge with each other due to capillary forces and other physical mechanisms. It would also be possible to dispense as a continuous flow stream or strip.

In embodiments of the invention, various pipettes 2020 may be aimed at corresponding target locations within web 300, which may differ in nature or local geometry from one pipette 2020 to another pipette 2020. For example, one pipette 2020 may be aimed at a location that is a crossover point where a fiber in one layer contacts a fiber in an adjacent layer, while another pipette 2020 may be aimed at a location that is not a crossover point. Thus, in various pipettes 2020, there may be some variation or randomness in the local nature of the screen geometry of the various droplets landing on the screen 300. Alternatively, if the spacing between pipettes 2020 is an integer multiple of the repeat size of the fibers in mesh panel 300, it would be possible for all pipettes 2020 to target mesh panel locations with the same geometric properties. The amount of fluid deposited by pipette 2020 at a location may be large enough to contain a large number of fibers 500, with the result that the location details of the deposited droplets do not make a significant difference in the deposition and spread of the fluid with respect to the fibers or spaces between the fibers.

In embodiments of the invention, it has been found that due to design trade-offs (trade off fs) and experiments with an attempt to provide the desired amount of open space in which cells can grow, it may be desirable for the spacing between fibers to be slightly larger than the diameter of an individual fiber. Adjacent layers may alternate with each other in their fiber direction. In this case, if the layers with fibres parallel to the fibres in the other layer are offset from each other, the number of open space see-through spaces will be reduced and better help to retain the liquid (medium and cells) deposited on the well plate. It has been found desirable if the maximum dimension of the see-through region is still smaller than the fiber diameter. This can be achieved as shown in fig. 5E to 5H. It has been found that this provides an acceptably small risk of sowing liquid (medium and cells) dripping through the mesh plate and failing to sow. Another parameter related to seeding is the volume of fluid deposited compared to the available free volume of the unit cell. It may be desirable to dispense an amount of fluid onto the web during sowing so that the dispensed fluid can be absorbed into or stored in the web, but cannot drip out of the web. For example, if the dispensers are arranged in a rectangular array at the spacing of typical wells in a microtiter plate (9 mm pitch in a square array), the unit volume of a well plate will be a 9mm by 9mm space. In another dimension, if the web comprises four layers of fibers, each layer having a diameter of 150 microns, the total height of the web is 0.6 mm. The unit volume is 9mm x 0.6mm or 48.6mm ^ 3. It was found by geometric calculations that if the fiber diameter was 150 microns and the spacing between opposing fiber edges (confronting edges) was 200 microns, the fraction of space occupied by the fibers was 34% and the fraction of space occupied by the empty spaces was 66%. Thus, in a cell, the empty space is 66% of 48.6mm ^3, or 32mm ^ 3. It was found experimentally that the cell had a good dispensed liquid volume of 15mm 3, while 23mm 3 of dispensed liquid was excessive for the cell (resulting in dripping from the well plate etc.). These dispensed fluid volumes can be compared to the void volume of the cell 32mm ^3 to show that satisfactory deposition occurred with 47% void volume in the cell, but if the occupancy of void volume in the cell is 72%, then an excess amount of fluid is indicated. Thus, a suitable operating range may be 40% to 70%, or more particularly 45% to 65%, of the void volume occupied in the unit cell.

In embodiments of the invention, it is possible to seed cells at a density of about 1200 to 5000 cells/cm 2 of the surface area of the mesh plate (referring to the curved surface area of all individual fibers in the mesh plate). It is believed that a seeding efficiency of about 90% can be achieved (i.e., of the cells deposited during seeding, about 90% of the cells will remain attached to the scaffold).

The dispenser may be operated by an automated system and may be programmed. In embodiments of the invention, the pipette and other components of the system may be operated so as to deposit or distribute the fluid evenly throughout the well plate 300. This may provide for an even distribution of liquid and cells from place to place within the well plate 300, and from one well plate to another, and may provide for batch to batch repeatability through the automated process. This is an improvement over manual pipetting of liquids and cells onto a rack.

After seeding is completed, in order to perform cell culture, it is possible to move the seeded scaffold into a bioreactor and gradually submerge the seeded scaffold in a liquid culture medium of the bioreactor. Submerging may be performed by raising the liquid level in the area where the mesh plate holder 200 may be loaded. This may be accomplished by operating a pump in the bioreactor system. This can be done slowly enough to avoid dislodging the seeded cells. This can be done continuously or in steps.

After the cell culture is complete, harvesting is the process of detaching the cells from the scaffold after culture. Harvesting may include exposing the scaffold to mechanical vibrations while also exposing the cells to a specific chemical environment that promotes cell separation. Furthermore, the design of the loadable mesh plate holder 200 is such that the culture medium may flow through the open interior of the loadable mesh plate holder 200. Such flow may be in a generally upward direction through the open spaces of the mesh panels 300. This harvesting may be performed while the mesh panels 300 (15 such mesh panels as shown) are in place in the loadable mesh panel holder 200. In this method, the isolated cells can be carried up by the flow medium and out of the culture area.

Features relating to sterility

Any of a variety of features useful for maintaining sterility may be provided.

It is possible to ship the loadable mesh plate holder 200 and the storage mesh plate holder 900 and the mesh plates 300 contained in the loadable mesh plate holder 200 to the customer in a pre-sterilized state. These components may have been assembled with each other along with the seeding tray 1300, and all of these components may be contained within suitable packaging to maintain sterility. It is possible to assemble all these components together or after assembly with each other, to sterilize them together. Sterilization of any component or group of components may be performed by any known sterilization method, including but not limited to gamma sterilization, ethylene oxide sterilization, and heat sterilization. The components may be suitably packaged to maintain sterility, either before or after the sterilization process.

As discussed herein, the seeding tray 1300 may have a grip 1320. The grip 1320 may protrude outward from the rest of the seeding tray 1300 and may allow a user to grasp the grip 1320. As a result, the user can carry the seeding tray 1300 and the loadable mesh plate holder 200 as well as the storage mesh plate holder 900 and any mesh plate 300 contained therein while keeping the user's hand at a distance from those components.

Given the unpredictable orientation that can occur to cargo during transport, a stop 1400 as described herein can be provided. The stop 1400 may thereafter be discarded. The stop 1400 may be sterile and may be included in the assembly when the assembly is sterilized.

Similarly, a handle 1500 may be provided to assist in lifting and moving the loadable mesh panel holder 200 and mesh panel 300 contained therein. The handle 1500 may be provided in a sterile state and may be a single-use component. The handle 1500 may be packaged with the assembly as it is sterilized, or alternatively, it may be sterilized and packaged separately.

The pusher 1100 may also be a single-use component and may be provided in a sterile state.

Similarly, any other desired auxiliary components may also be provided under sterile conditions.

Further remarks

In other embodiments of the invention, it is possible that the pusher 1100 may still move in a generally horizontal direction as shown, but the pusher 1100 itself may have a different orientation, such as a vertical direction, to access the loadable mesh holder 200 from below.

In general, any combination of the disclosed features, components, and methods described herein is possible. Unless otherwise indicated, the steps of the methods may be performed in any order that is physically possible.

All cited references are incorporated herein by reference.

Although embodiments have been disclosed, it is not intended to be so limited. Rather, the scope of the invention should be determined from the following claims.

Claims

1. A system for creating a stack of cell-seeded well plates, the system comprising:

a plurality of net plates;

a storage screen holder adapted to receive the plurality of screens;

a loadable mesh panel holder adapted to receive the plurality of mesh panels;

a pusher adapted to push individual ones of the mesh panels from the storage mesh panel holder to the loadable mesh panel holder;

a dispenser adapted to dispense a liquid containing said cells onto one of said well plates,

wherein the storage screen holder and the loadable screen holder are arranged at a distance from each other such that one of the screens accommodated in the storage screen holder can be slid into the loadable screen holder in a planar translational movement; and

a motion control system adapted to control motion or position of at least some of the storage screen holder, the loadable screen holder, the pusher, the screen, and the dispenser.

2. The system of claim 1, wherein the loadable and storage screen holders include respective grooves that are collinear or coplanar with one another in which the screens can slide from one of the screen holders to another of the screen holders.

3. The system of claim 1, wherein one of the mesh panels can reside in the loadable mesh panel holder without extending outside an outer envelope of the loadable mesh panel holder, but when the mesh panel resides in the storage mesh panel holder, the mesh panel extends outside an outer envelope of the storage mesh panel holder.

4. The system of claim 1, wherein the pusher has a thickness in a vertical direction that is less than twice a separation distance of a central web to a central web in the vertical direction.

5. The system of claim 1, wherein the pusher is removably connected to the motion control system by a magnetic attachment.

6. The system of claim 1, further comprising a seeding tray, wherein the seeding tray and the storage screen holder and the loadable screen holder and the plurality of screens are assembled together to form an assembly, and the assembly is provided in a sterile state within a package adapted to maintain sterility while the assembly is within the package.

7. The system of claim 1, wherein the motion control system comprises: a first drive capable of moving the storage screen holder and the loadable screen holder in a first direction; a second driver capable of appropriately moving the pusher to slide one of the mesh plates in a second direction; and a third actuator capable of moving the dispenser.

8. The system of claim 1, wherein the motion control system comprises: a first horizontal drive system capable of moving the pusher in a first horizontal direction; and a second horizontal drive capable of moving the dispenser in a second horizontal direction perpendicular to the first horizontal direction, and a vertical drive capable of moving the storage screen holder and the loadable platen holder in a vertical direction.

9. The system of claim 1, further comprising a reservoir adapted to contain a fluid, wherein the motion control system comprises a vertical drive capable of changing the relative vertical position of the dispenser and the reservoir.

10. The system of claim 1, wherein the web comprises four layers, each layer comprising fibers that are substantially straight and substantially parallel to other of the fibers in the layer, the fibers being substantially perpendicular to fibers in an adjacent layer of the web, wherein the fibers in the layers are staggered in a first direction and further staggered in a second direction different from the first direction.

11. The system of claim 1, wherein the web comprises a plurality of intersecting fibers, wherein the fibers have a contact angle with pure water of less than 50 degrees.

12. A method for creating a stack of cell-seeded mesh plates, the method comprising the steps of:

providing the system of claim 1;

operating the system such that a lowermost one of the plurality of well plates in the storage well plate holder, a first moving well plate, is moved into the loadable well plate holder and then dispensing fluid from the dispenser onto the first moving well plate, followed by moving a next higher well plate from the storage well plate holder into the loadable well plate holder and followed by dispensing fluid onto the next higher well plate.

13. The method of claim 12, wherein the system further comprises a reservoir containing a quantity of the fluid, and further comprising, prior to dispensing the fluid from the dispenser, drawing some of the fluid into the dispenser and thereafter ejecting at least some of the drawn fluid into the reservoir, and then drawing some of the fluid into the dispenser.

14. The method of claim 12, wherein a flow rate during said drawing of said fluid into said dispenser is different from said ejecting of said fluid into said reservoir.

15. The method of claim 12, wherein the first and second light sources are selected from the group consisting of,

wherein the dispensers define a unit cell having a planar area of the web associated with an individual one of the plurality of dispensers, wherein the unit cell further has a vertical dimension that is a distance between a top of the web and a bottom of the web, wherein the unit cell has a total volume equal to the planar area multiplied by the vertical dimension, wherein the unit cell further has a solid volume occupied by fibers within the unit cell, wherein the unit cell further has an empty volume that is the total volume minus the solid volume;

dispensing a fluid onto the web from an individual one of the plurality of dispensers in a vertically downward direction substantially perpendicular to the web, wherein a dispensed volume of the fluid dispensed by an individual one of the plurality of dispensers fills 40% to 70% of an open volume of the unit cell of the web.