WO2019168838A1

WO2019168838A1 - Scaffold loader for bioreactor

Info

Publication number: WO2019168838A1
Application number: PCT/US2019/019565
Authority: WO
Inventors: Zongsen Wang; Wing Lau; Faribourz Payvandi
Original assignee: 3D Biotek, Llc
Priority date: 2018-02-27
Filing date: 2019-02-26
Publication date: 2019-09-06
Also published as: CN111770987A

Abstract

A system for seeding cells onto an array of screens or scaffolds is provided. The system can include a storage screen holder and a loadable screen holder, which can be in spaced relationship such that screens can move from the storage screen holder into the loadable screen holder, when urged by a pusher mechanism. Cells for seeding can be dispensed by a dispenser. A motion control system can move appropriate ones of the screens, screen holders and dispenser as needed. The lowest screen can be moved into the loadable screen holder, and cells can be dispensed onto it, and then successively higher screens can be moved into the loadable screen holder and seeded with cells. Components such as the storage screen holder the loadable screen holder and the screens can be supplied as a pre-sterilized assembly.

Description

Scaffold loader for bioreactor

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

62/636,039 filed February 27, 2018, the disclosure of which is hereby incorporated by reference herein in its entirety.

Field of the Invention

[0002] Embodiments of the invention pertain to bioreactors.

Background of the Invention

[0003] Bioreactors are used to expand a population of cells, such as stem cells or other anchorage dependent cells. However, improvements are still desirable, such as in regard to ease of use, and automation, and reproducibility of procedures. It is desirable to culture so as to produce large numbers of cells, such as billions of cells if possible. Seeding of scaffolds is an area in which improvement would be useful. Various bioreactors and components thereof 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 herein in their entireties.

Brief Description of the Invention

[0004] In an embodiment of the invention, there may be provided a system for creating a stack of screens that are seeded with cells, the system comprising: a plurality of screens; a storage screen holder suitable to hold the plurality of screens; a loadable screen holder suitable to hold the plurality of screens; a pusher suitable to urge individual ones of the screens from the storage screen holder to the loadable screen holder; a dispenser suitable to dispense liquid containing the cells onto the screens; wherein the storage screen holder and the loadable screen holder are in spaced relation to each other so that one of the screens held in the storage screen holder can be slid in a planar translation motion into the loadable screen holder; and a motion control system suitable to control motion or position of at least some of the storage screen holder, the loadable screen holder, the pusher, the screens and the dispenser.

[0005] An embodiment of the invention may comprise a method for creating a stack of screens that are seeded with cells, the method comprising the steps of: providing a storage screen holder; providing a plurality of screens, the screens being unseeded; loading the plurality of screens into the storage screen holder, the screens being loaded one above another; providing a loadable screen holder; assembling the storage screen holder and the loadable screen holder in relation to each other to form an assembly, the assembly being configured such that any one of the screens in the storage screen holder can slide into a corresponding position in the loadable screen holder; installing the assembly onto a machine comprising a motion control system and a dispenser; and operating the machine so that a lowest one of the screens in the storage screen holder is moved into the loadable screen holder being a first-moved screen, and then liquid is dispensed from the dispenser onto the first-moved screen, followed by moving a next-higher screen from the storage screen holder into the loadable screen holder, followed by dispensing liquid from the dispenser onto the next-higher screen.

[0006] An embodiment of the invention may comprise a method for creating a screen that is seeded with cells, the method comprising the steps of: providing a screen, the screen being unseeded, wherein the screen comprises four layers, each layer comprising fibers that are generally straight and generally parallel to others of the fibers in the layer, the fibers being generally perpendicular to fibers in an adjacent layer of the screen, the fibers within a layer being separated from adjacent fibers within the layer by a separation distance that is greater than a fiber diameter of the fiber, wherein the screen in plan view has see-through regions whose largest dimension is smaller than the fiber diameter, wherein the fibers have a contact angle with pure water that is less than 50 degrees; providing a plurality of liquid dispensers arranged in a regular array, wherein the dispensers define a unit cell having a plan area of the screen associated with an individual one of the dispensers, wherein the unit cell further has a vertical dimension being a distance between a top of the screen and a bottom of the screen, wherein the unit cell has a total volume equal to the plan 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 liquid from the dispensers onto the screen, in a vertically downward direction generally perpendicular to the screen, wherein a volume of the dispensed liquid fills between 40% and 60% of the open volume of the screen.

Brief Description of the Illustrations

[0007] Embodiments of the invention are further described but are in no way limited by

the following illustrations: [0008] Figure 1 is a three-dimensional view showing a loadable screen holder, without screens;

[0009] Figure 2 is a three-dimensional view showing the loadable screen holder of Figure

1, exploded;

[0010] Figure 3 A is a three-dimensional view showing the loadable screen holder with screens in it;

[0011] Figure 3B is a three-dimensional view showing the loadable screen holder of

Figure 3A with one part of the loadable screen holder missing in order to better show the screens;

[0012] Figure 3C is a three-dimensional view showing the loadable screen holder of

Figure 3B with some of the screens missing;

[0013] Figure 4A is a view of the closure piece of the loadable screen holder, showing rounded entrances of slots in the loadable screen holder;

[0014] Figure 4B is another view of the closure piece of the loadable screen holder, from a different perspective;

[0015] Figure 4C is a view of a local region of the closure piece with screens present;

[0016] Figure 5 A shows a screen made according to an embodiment of the invention;

[0017] Figure 5B shows a closer view of a similar screen;

[0018] Figure 5C illustrates spacing parameters of the screens such as those in Figures

5A and 5B;

[0019] Figure 5D illustrates more specifically the placement of fibers within a screen, and their staggering;

[0020] Figure 5E shows a plan view of just one layer of a screen;

[0021] Figure 5F shows a plan view of just two layers of a screen;

[0022] Figure 5G shows a plan view of just three layers of a screen;

[0023] Figure 5H shows a plan view of four layers of a screen;

[0024] Figure 51 illustrates a screen having a thickness that is nonuniform;

[0025] Figure 6 illustrates a possible leakage flowpath; [0026] Figure 7A illustrates the three-sided piece that is part of the loadable screen holder, so as to illustrate positioning slots;

[0027] Figure 7B is a cutaway view illustrating the three-sided piece of Figure 7A, together with a pusher accessory;

[0028] Figure 7C illustrates the pusher accessory;

[0029] Figure 8A is a three-dimensional illustration of the storage screen holder;

[0030] Figure 8B is another three-dimensional illustration of the storage screen holder, also showing several screens;

[0031] Figure 9A is a three-dimensional illustration of the storage screen holder and the loadable screen holder positioned in their side-by-side position;

[0032] Figure 9B is a cross-section of Figure 9A, also showing one screen;

[0033] Figure 10A is a three-dimensional illustration of the storage screen holder and the loadable screen holder exploded away from each other, for clarity of illustration;

[0034] Figure 10B is a cross-section of Figure 10A;

[0035] Figure 11 A is a three-dimensional illustration of a seeding tray;

[0036] Figure 11B is a three-dimensional illustration showing the storage screen holder and the loadable screen holder side-by-side in the seeding tray that supports and locates them;

[0037] Figure 12A is a three-dimensional view of the storage screen holder and the loadable screen holder and a stop suitable to prevent motion of screens during shipping;

[0038] Figure 12B is a three-dimensional view of a lifting handle in isolation;

[0039] Figure 12C is a three-dimensional view of the lifting handle engaged with a loadable screen holder;

[0040] Figure 13A is an exploded view illustrating the pusher and its support;

[0041] Figure 13B is similar to Figure 13A from a different perspective;

[0042] Figure 14A is a three-dimensional view of the storage screen holder and the loadable screen holder and a dispenser for dispensing cells for seeding onto screens;

[0043] Figure 14B is similar to Figure 14A except from a different perspective; [0044] Figure 14C is a cross-section of Figure 14B; and

[0045] Figure 15 is a three-dimensional frontal view of the entire system or machine.

Detailed Description of the Invention

[0046] Reference is now made to Figures 1-7C. As described in US patent application Serial

Number 15/686,211, in an embodiment of the invention there may be provided a bioreactor having a loadable screen holder 200 suitable to hold a plurality of screens 300. In a bioreactor, cells may reside and grow upon screens 300. The array of screens 300 can be referred to as a culture structure.

[0047] Loadable screen holder 200 is illustrated in Figures 1-4C. Loadable screen holder 200 may hold a desired number of screens 300 in a desired spaced position and orientation. The loadable screen holder 200 illustrated in Figure 1-4C holds 15 screens 300. This is believed to be an appropriate number of screens for operation of the bioreactor.

[0048] In general, loadable screen holder 200 may surround an interior space and may be any of various shapes such as round, rectangular, etc. As illustrated, the loadable screen holder 200 (on both its both interior and its exterior) has the general shape of a rectangle with rounded comers. The screens 300 held by the illustrated loadable screen holder 200 are rectangular. For sake of convenience in description, loadable screen holder 200 is described here using directional designations such as front and back, sides, and horizontal and vertical, which generally correspond to their orientation or position as illustrated in the assembled bioreactor. However, it may be understood that these directional designations are somewhat arbitrary.

[0049] When screens 300 are in place in loadable screen holder 200, there may be a desired amount of space in the vertical direction between a screen 300 and its nearest neighbor screen. In the bioreactor as illustrated, the flow of the liquid medium can be generally perpendicular to the flat surface of screens 300, flowing through the open spaces in the screen 300.

[0050] A cell culture region could contain, for example, approximately 15 such screens 300 spaced apart from each other by a sufficient distance so that the screens do not touch each other (resulting from possible deflection or bowing of the screens or any other reason), and some sideways flow of liquid medium in the space between adjacent screens 300 is possible if necessary. [0051] Loadable screen holder 200 could be of a hollow simple shape some of which forms a complete perimeter, with the shape being a shape such as round or rectangular (possibly with rounded comers). In general, it is possible that loadable screen holder 200 could be made as a single piece, such as by an additive manufacturing process. However, perhaps more typically loadable screen holder 200 could be made out of two pieces, such as two pieces of molded plastic, that join together with each other. It is illustrated that loadable screen holder 200 is made of two inter-engaging pieces. The loadable screen holder 200 may comprise a three- sided piece 220 and a closure piece 240 that is engageable with the three-sided piece 220. The three-sided piece 220 may comprise, in sequence, a first side segment 222A, a rear segment 222B and a second side segment 222C opposed to the first side segment 222A. The closure piece 240 may comprise at least one segment, which may be considered a front segment 242B. As illustrated, the closure piece 240 may additionally comprise a first side segment 242A and a second side segment 242C. Thus, in sequence, there is a first side segment 242A, a front segment 242B and a second side segment 242C. The first side segment 242A may be engageable with the first side segment 222A, and the second side segment 242C may be engageable with the second side segment 222C. However, it is also possible that the closure piece 240 might comprise only a front segment 242B. As illustrated, front segment 242B and rear segment 222B are substantially parallel to each other, and first side segment 222A and second side segment 222C are substantially parallel to each other and first side segment 242A and second side segment 242C are substantially parallel to each other. However, other spatial relationships are also possible.

[0052] The engagement features may comprise deformable tabs 250, and further may comprise an opening 224A in first side segment 222A and a similar opening 224C in second side segment 222C, with the openings 224A and 224C being appropriately designed to engage with tabs 250. The tabs 250 may be elastically deformable between an engagement position as illustrated, and a release position in which the deformable tabs 250 are bent inward sufficiently to create disengagement. The tabs 250 may comprise living hinges, and it is further possible that elasticity elsewhere in the closure piece 240 may also contribute to changes of dimension or shape so as to permit or assist in achieving disengagement.

[0053] Loadable screen holder 200 may comprise mechanical interface features, illustrated as holes 260, which may be located near the top of loadable screen holder 200 in the illustrated orientation. Such holes 260 may be appropriate to interface with a lifting handle as described elsewhere herein. [0054] Loadable screen holder 200 may have an upper edge and a lower edge that may be flat, and which may be parallel to each other.

Slots and grooves in loadable screen holder, and rounded edges near slots and grooves

[0055] The loadable screen holder 200 may comprise various grooves and slots that define locations of screens 300 and provide mechanical support for screens 300. As illustrated, loadable screen holder 200 is able to hold 15 of the screens 300, but of course other numbers of screens 300 are also possible. The spacing distance between screens 300 may be chosen from a combination of considerations such as patterns of fluid flow between screens 300, and desired overall packing density of cells in the cell culture region of bioreactor.

[0056] If loadable screen holder 200 were made as a single piece (similar to what is illustrated but with the three-sided piece 220 and the closure piece 240 joined together), it would be possible to insert and remove screens 200 into or from loadable screen holder 200. This can be done from a sideways direction.

[0057] In the loadable screen holder 200 as described and illustrated, it is possible to insert and remove screens 300 into or from loadable screen holder 200 when three-sided piece 220 and closure piece 240 are already assembled to each other, without disassembling those pieces from each other. This can be done from a sideways direction. As illustrated, first side segment 222A has grooves 270, and second side segment 222C has grooves 270. Rear segment 222B has grooves 270. Front segment 242B has slots through which screens 300 may pass without disassembly of loadable screen holder 200. The grooves 270 may be substantially co-planar with each other and may have similar or identical dimensions to each other in the vertical direction. Slots 280 may be parallel to or coplanar with at least some of grooves 270. The vertical dimensions and elevations of slots 280 may be identical to those of grooves 270, although it is sufficient if the vertical dimensions and elevations are simply generally similar to each other. These various features may combine and interact so that when loadable screen holder 200 is assembled, it is possible for screens 300 to be slid into place through slot 280 and to be supported by grooves 270, as well as by slot 280. The desired spacing or separation between screens 300 is maintained by the material of loadable screen holder 200 near grooves 270, and by the material of loadable screen holder 200 (ligaments) that exist between slots 280.

[0058] As illustrated, closure piece 240 has three sides, with two of the sides 242A, 242C being short. One side wall of loadable screen holder 200 comprises both first side segment 222A and first side segment 242A, and similarly the other side wall comprises both second side segment 222C and second side segment 242C. As illustrated, even tabs 250 contain grooves that are continuous with grooves 270 in the corresponding adjacent side segments. However, it can be understood that various other designs are also possible.

[0059] As illustrated, grooves 270 in first side segment 222A and first side segment 242A are substantially sharp-edged. The same is true for grooves 270 in second side segment 222C and second side segment 242C. However, it can be understood that other geometries are also possible.

[0060] On a front portion of the loadable screen holder 200, the separators or ligaments of remaining material may be such that there are rounded edges face generally exteriorly of the loadable screen holder 200. This can be helpful in guiding screens 300 into slots 280 during initial insertion of the screens 300 into slots 280. The rounded edges may be hemicylindrical. Such rounded edges can also be helpful in guiding screens 300 into slots 280 if it happens that any of the screens 300 are not perfectly planar, such as being slightly warped out-of-plane.

[0061] At a rear portion of the loadable screen holder 200, separators between individual screens 300 may be such that there are rounded edges face generally toward the interior of the loadable screen holder 200. This can be helpful in guiding screens 300 into desired positions when screens 300 are being inserted into loadable screen holder 200, such as near the end of the insertion process. The rounded edges may be hemicylindrical. The rounded edges may be hemicylindrical. Such rounded edges can also be helpful in guiding screens 300 into grooves 270 if it happens that any of the screens 300 are not perfectly planar, such as being slightly warped out-of-plane.

[0062] The separators at the front portion of the loadable screen holder 200 may define slots through which the screens 300 may pass. The separators at the rear portion of the loadable screen holder 200 may define grooves into which the screens 300 may enter but through which it is impossible for screens 300 to pass.

[0063] In the case of both the rounded edges of the slots 280 and the rounded edges of grooves

270, a hemicylindrical curvature is only one of various possible curvatures. A fillet having some other desired radius is also possible. Such radius could be either less than or greater than the radius of the hemi cylinder. Other shapes of curves are also possible. Variation of curvature along the length of the slot 280 or groove 270 is also possible. Design of screens

[0064] In embodiments of the invention, and referring now to Figures 5A-5H, the bioreactor may have, within the culture region, an array of screens 300, which serve as tissue scaffolds for cells such as anchorage-dependent cells to grow upon. The individual screens 300 themselves may comprise a number of layers of fibers 500, with the fibers 500 having orientations that are perpendicular to the orientations of fibers in an adjacent layer. In a particular embodiment of the invention, the number of layers of such fibers 500 in an individual screen 300 may, for example, be four or five or six layers.

[0065] Typical dimensional parameters of such screens 300 could be a fiber diameter of 150 microns and a fiber spacing (as defined in Figure 5C) of 200 microns, with that dimension referring to the distance from an edge of one fiber to the nearest edge of the nearest fiber in the same plane. Fibers 500 in one layer may be staggered relative to fibers 500 that are parallel to them and are located in a different layer of the screen 300, or alternatively there does not have to be staggering. The fibers 500 may be spaced apart from each other by appropriate distances such that during initial cell seeding, the cells deposit on fibers 500 and do not touch cells on adjacent fibers 500. However, the spacing of the fibers 500 may be such that after some number of cell multiplications, cells growing on nearby fibers 500 may contact or grow against each other (a situation known as confluence).

[0066] More particularly, the fibers 500 may exist in two mutually perpendicular directions and may be staggered in both of those two mutually perpendicular directions. This is further illustrated in Figure 5D. Each screen may comprise a plurality of fibers forming a first layer having parallel fibers oriented in a first direction and may comprise a second layer having a plurality of parallel fibers oriented in a second direction that is perpendicular to the first direction, the fibers in the second layer being joined to the fibers in the first layer. At least some of the screens comprise a plurality of fibers, and within an individual one of the screens, the fibers are arranged in sequence in at least first, second, third and fourth layers, the fibers within each of the layers being generally parallel to each other, wherein fibers in the first layer are generally parallel to fibers in the third layer and fibers in the second layer are generally parallel to fibers in the fourth layer, and wherein when viewed perpendicular to a flat surface of the screen, fibers of the third layer are located non-aligned with fibers of the first layer and fibers of the fourth layer are located non-aligned with fibers of the second layer. More specifically, with respect to this viewing direction, the fibers may be located midway between the parallel fibers that they are not aligned with. Such configuration can help to prevent cells from falling through the screen, especially during initial seeding, while still providing space for liquid medium to occupy and flow through, and providing space for cells to grow into as the cells multiply.

[0067] A screen 300 may have a particular number of layers, such as four layers, with media available at both surfaces of the four-layer construct, for contacting cells, and media also available in openings that exist between layers. It is believed that all of the features of this situation more closely resemble what occurs in natural tissues, which can be termed a three- dimensional environment. It is believed that the three-dimensional situation of embodiments of the invention is more conducive to cell multiplication and expansion than is a two- dimensional environment. However, it is not wished to be limited to this explanation.

[0068] Referring now to Figures 5E-5H, there is shown a plan view of the screen of Figures

5A-5D, with varying numbers of layers being shown. (The showing of less than all of the layers of fibers is simply done for clarity of illustration.) In Figure 5E, only one layer is shown. Figure 5F shows that first layer and a second layer whose fiber orientation is perpendicular to that of the layer shown in Figure 5E. Figure 5G adds a third layer, whose fiber orientation is parallel to that of the first layer and whose fiber locations are staggered with respect to the first layer. It can be observed that there is not a lot of see-through, and the width of the see-through regions is smaller than a fiber diameter. Finally, Figure 5H shows four layers, with the newly- shown fourth layer having fibers that are parallel to those of the second layer and staggered with respect to it. It can be observed that the sizes of the see-through regions are even smaller than what was visible in Figure 5G, and the dimensions of the see-through regions are smaller than a fiber diameter. It is believed that the small size of the see-through regions, relative to the fiber diameter, is helpful for retaining liquid that may be deposited on the screens during seeding.

[0069] In another embodiment of the invention, there might be provided a screen 300 that contains six layers of filaments. Just as in the illustrated four-layer screen, adjacent layers may have fiber orientations that are perpendicular to each other. However, the use of six layers provides more possible choices as far as offsets between layers that have the same fiber orientation. For example, the offset distance between one layer and another layer having the same fiber orientation could be one-third of the inter-fiber spacing, and the offset between that one layer and yet another layer having the same fiber orientation could be one-third of the inter- fiber spacing in the opposite direction. For example, using the same fiber diameter and inter- fiber spacing as already illustrated, and the just-described offsets, it would be possible to construct such a six-layer screen in which there is no line-of-sight pathway (see-through) in a direction perpendicular to the overall surface of the screen.

[0070] The screens 300 may be formed by a programmed deposition of heated filaments of polymer, similar to what is described in commonly-owned US patent 8,463,418. The polymer may be a suitable biocompatible polymer such as polystyrene. The screen 300 as described is non-woven. Alternatively, screen 300 may be woven if desired. The overall shape of the screen 300 may be flat and rectangular.

[0071] Referring now to Figure 51, it is further possible that a screen 300 could have a thickness that is nonuniform. For example, the thickness of the screen 300 near the edges, where the screen 300 slides in grooves, could be relatively thinner, and the thickness of the screen 300 away from its edges could be relatively thicker. The thicker central portion could provide more space for holding more cells during culturing, compared to the situation of a uniform-thickness screen 300.

[0072] In some prior art cell culturing techniques such as Petri dishes, a layer of cells grows on a flat surface and experiences an environment that is essentially two-dimensional. Even if there is adequate supply of nutrients and removal of waste products in this situation, such a two-dimensional environment is by inherently different from the environment in which cells naturally grow, which is a three-dimensional environment.

[0073] In embodiments of the invention, cells seed by attaching onto individual fibers of the screen. The fiber-to-fiber dimension of the screen may be large enough that when isolated cells initially attach to the fibers, at least some of the cells generally do not touch other cells. After cells have multiplied, and perhaps created several layers of cells where initially only one layer of cells was attached to a fiber, it is possible that the outermost cells may still be independent of the next fiber or it is possible that some of the new cells may touch other cells that are attached to other fibers, i.e., some bridging of fibers may occur (referred to as confluence). In embodiments of the invention, a screen may have a particular number of layers of fibers, such as four layers with media available at both surfaces of the four-layer construct, for contacting cells. At any rate, it is believed that all of the features of this situation more closely resemble what occurs in natural tissues, which can be termed a three-dimensional environment. It is believed that the three-dimensional situation of embodiments of the invention is more conducive to cell multiplication and expansion than is a two-dimensional environment. However, it is not wished to be limited to this explanation. [0074] A cell culture region could contain, for example, approximately 10 to 15 such screens spaced apart from each other by a sufficient distance so that the screens do not touch each other and liquid can flow between the screens.

[0075] In terms of biological parameters, a screen used with an embodiment of the invention may have an area (length dimension * width dimension) of about 7000 mm^A2, and the portion of that screen exposed to perfusion (excluding the edges that are in grooves or slots) may have an area of about 6300 mm^A2. This screen may be formed of four layers of fibers, with the layers alternating in direction as described elsewhere herein. Alternatively, it is possible to use five or six layers of fibers or some other number. On such a screen there may be deposited an initial seeding of about 800,000 cells, so that if 12 screens are used, the population of seeded cells is 9.6 million cells. At the end of culturing, that cell population may be expanded by a factor of about 25.

[0076] For purposes of culturing cells and for the seeding of cells onto scaffolds, it is believed to be desirable for the surface properties of the scaffold material to be hydrophilic. Some polymers that are useful scaffold materials are not inherently as hydrophilic as might be desired. In general, it may be desirable that a drop of water or aqueous liquid spread upon and within the screen after being deposited onto the screen. In embodiments of the invention, after the screen has been created by appropriate positioning of fibers, the polymeric scaffold material such as polystyrene may be subjected to a plasma treatment. Plasma treatment may be done in a plasma chamber apparatus such as is available from Harrick Plasma (Ithaca, NY). In such an apparatus, gas at a low sub-atmospheric pressure is subjected to radiofrequency electromagnetic radiation creating plasma at near ambient temperatures within a treatment chamber. Gases used can be argon, oxygen, air, hydrogen, nitrogen, mixtures of any of these, or other choices, or no special composition of gas need be added at all. Such a treatment can clean the surface. Such a treatment can accomplish surface modification, which can include changing the surface energy of the surface in either direction depending on details of the treatment. The surface modification can involve attaching atoms or functional groups, such as functional groups containing oxygen, onto the polymer. The surface treatment can result in the surface of the polymer becoming more hydrophilic than it was prior to the treatment. For example, the surface properties of the polymer surface after treatment can 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 condition, and a larger contact angle indicates a more hydrophobic condition.) After the plasma treatment, it may also be appropriate to perform a static charge removal process at the end of the plasma treatment. Such a treatment can remove or neutralize any static charge that may remain at the end of the plasma treatment. Such a treatment can be performed using a static eliminator from Keyence Corporation (Itasca, IL). Such a device discharges a stream containing both positively charged ions and negatively charged ions toward a workpiece, and whichever ions are needed to neutralize static charge contact the surface and neutralize the static charge.

Pusher accessory and slots for positioning of screens

[0077] Referring now to Figure 7A-7C, in embodiments of the invention, as described herein, the loadable screen holder 200 may comprise positioning slots 390 that can be used for pushing screens 300 into a desired position or for defining positions of screens 300. Such positioning slots 390 may be generally vertical with respect to overall directions of the loadable screen holder 200 and bioreactor. Positioning slots 390 may intersect other slots and grooves that are provided in loadable screen holder 200.

[0078] A pusher accessory 400 may also be provided as an accessory (for use before or after actual culturing). It is possible that the pusher accessory 400 can be used on one side of the loadable screen holder 200 for the purpose of pushing screens 300 into loadable screen holder 200 until they contact a stop such as the base of groove 270. It is also possible that pusher accessory 400 may be used on an opposite side of loadable screen holder 200 for the purpose of pushing screens 300 out of loadable screen holder 200, such as when culturing is completed or it is desired to remove screens 300. Positioning slots 390 on one side of loadable screen holder 200 may have identical or similar dimensions and spacing as positioning slots 390 on another side of loadable screen holder 200, which would enable a single pusher accessory 400 to be used for pushing in both directions.

[0079] In order to facilitate the described pushing, pusher accessory 400 may have certain dimensional relationships with appropriate features of loadable screen holder 200. Bosses 420 on pusher accessory 400 may be dimensioned, and may be spaced appropriately with respect to each other, so that they can fit into positioning slots 390 of loadable screen holder 200. The height of the bosses 420 on pusher accessory 400 may be sufficiently large so that the screens 300 may be pushed to a desired extent.

Storage screen holder [0080] Referring now to Figures 8A and 8B, in addition to providing loadable screen holder

200, there may also be provided a storage screen holder 900. Storage screen holder 900 may have some geometric similarities to loadable screen holder 200 but also may be different in some respects.

[0081] Storage screen holder 900 may be have dimensions and features appropriate to hold screens 300 in a geometry such that screens 300 in storage screen holder 900 are coplanar with their eventual respective positions in loadable screen holder 200. Specifically, storage screen holder 900 may have internal grooves 906 suitable to receive screens 300. Such grooves may be dimensioned and located similarly or identically to corresponding features in loadable screen holder 200. As illustrated, the grooves 906 on the side of storage screen holder 900 through which screens would be loaded into storage screen holder 900, have rounded internal edges, which may help screens 300 to enter those grooves.

[0082] Storage screen holder 900 may have an opening 910 that is suitable to allow a pusher

1100 to occupy space when necessary and to contact a trailing edge of a screen 300 and urge screen 300 out of storage screen holder 900 into loadable screen holder 200. Opening 910 may be dimensioned and located such that pusher 1100 never actually contacts any of the boundaries of opening 910 when pusher 1100 is in any of its allowed locations.

[0083] The side of storage screen holder 900 that is intended to abut loadable screen holder

200 is shown as being unobstructed. That may make it easier for screens 300 to slide out of storage screen holder 900 into loadable screen holder 200. Such empty space allows the pusher 1100 to push screens 300 into loadable screen holder 200 until pusher 1100 approaches or makes contact with the corresponding surface of recess 210. This helps to achieve desired positioning of screens 300 in loadable screen holder 200.

[0084] As illustrated, storage screen holder 900 has a top surface 920, which may contribute to the structural connectedness of storage screen holder 900. Of course, variations of these design features are possible.

[0085] Figure 8B shows several screens 300 in place in storage screen holder 900. As illustrated, the dimensional relationship between the width of screens 300 and the width of storage holder 900 is such that screens 300 extend beyond storage screen holder 900, to an extent that screens 300 would occupy a slight amount of space in loadable screen holder 200 when loadable screen holder 200 and storage screen holder 900 are in their operational relative configuration. As a result, at the time when pusher 1100 tries to advance a screen 300, the leading edge of that screen 300 would already have passed the interface between storage screen holder 900 and loadable screen holder 200, and this would eliminate any possibility of screen 300 becoming mechanically stuck at the interface between storage screen holder 900 and loadable screen holder 200.

Assembly of storage screen holder and loadable screen holder

[0086] Referring now to Figures 9A, 9B, 10A and 10B, there is illustrated both storage screen holder 900 and loadable screen holder 200. Figures 9A, 9B show storage screen holder 900 and loadable screen holder 200 in the relation to each other when assembled for use. Figures 10A, 10B show storage screen holder 900 and loadable screen holder 200 exploded away from each other.

[0087] Storage screen holder 900 may have appropriate dimensions and features so that storage screen holder 900 can be aligned with loadable screen holder 200 to achieve the relationship that screens 300 in storage screen holder 900 are coplanar with their eventual respective positions in loadable screen holder 200. For example, some of the external dimensions of storage screen holder 900 and of loadable screen holder 200 may be identical to each other in order to facilitate such alignment, and internal dimensions regarding vertical positioning of the screens 300, such as dimensions and positions of grooves and slots, 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 connecting storage screen holder 900 and loadable screen holder 200 directly to each other, or similar features known in the art, although such are not illustrated herein. It would also be possible to use a seeding tray for alignment as discussed herein. The dimensional and other relationships between the screens 300 and the grooves in storage screen holder 900 and loadable screen holder 200 may be such that the fit of the screens in the grooves is loose enough so that the screens 300 can be easily slid between storage screen holder 900 and loadable screen holder 200 without requiring application of a large amount of force.

[0088] As illustrated, storage screen holder 900 has in one side an opening 910 suitable for pusher 1100 to pass through.

[0089] The spacing, in the vertical direction, between grooves in storage screen holder 900 and loadable screen holder 200, and the vertical positioning of those grooves, may be identical to each other, so that screens 300 can easily slide from grooves in storage screen holder 900 into grooves in loadable screen holder 200.

[0090] Figure 10A (full view) and Figure 10B (sectioned) show storage screen holder 900 and loadable screen holder 200, similar to Figures 9A and 9B, except that storage screen holder 900 and loadable screen holder 200 are separated from each other for clarity of illustration.

Seeding tray

[0091] Referring now to Figure 11 A and Figure 11B, as illustrated, the lower portion of storage screen holder 900 and the lower portion of loadable screen holder 200 may both be contained in a closely-fitting seeding tray 1300. The sides of seeding tray 1300 may abut respective sides of storage screen holder 900 and the lower portion of loadable screen holder 200 and thereby provide geometric alignment of those screen holders with each other. Such fits and relationships may also provide registration of the positions of storage screen holder 900 and loadable screen holder 200 relative to each other and to other components of the machine.

[0092] Seeding tray 1300 may have, in its side, open space 1310 appropriate to accommodate all desired positions of pusher 1100, such as to allow pusher 1100 to pass through the side of seeding tray 1300 in all of the allowed positions of pusher 1100. Open space may also be provided for any components located near or attached to pusher 1100. In other designs, seeding tray 1300 may have appropriate openings in its bottom to accommodate all desired positions of pusher 1100.

[0093] Seeding tray 1300 may have a gripping handle 1320 protruding therefrom. Gripping handle 1320 may be such that a user can grasp gripping handle 1320 and thereby carry seeding tray 1300 and loadable screen holder 200 and storage screen holder 900 and any screens 300 contained therein, while the user’s hand remains at some distance from those components. This may aid in maintaining sterility of those components during handling of those components after they have been removed from their sterile packaging.

[0094] Seeding tray 1300 may be configured and dimensioned such that it aligns storage screen holder 900 and loadable screen holder 200 in desired direction, but even when storage screen holder 900 and loadable screen holder 200 are in seeding ray 1300, it is permitted that the loaded and seeded loadable screen holder 200 is able to be vertically lifted out. For example, seeding tray 1300 may be such that the bottom surfaces of storage screen holder 900 and loadable screen holder 200 are coplanar with each other, and one side of of storage screen holder 900 is in contact with a corresponding side of loadable screen holder 200, and one side of storage screen holder 900 is coplanar with one side of loadable screen holder 200, and an opposed side of storage screen holder 900 is coplanar with a corresponding side of loadable screen holder 200.

[0095] It is possible that loadable screen holder 200 and storage screen holder 900 may be vertically slidable with respect to each other, in the sense that if loadable screen holder 200 and storage screen holder 900 are already present in the seeding tray 1300, it is possible or loadable screen holder 200 to be slid into or out of place in seeding tray 1300, using a generally vertical motion. This can be possible if seeding tray 1300 already provides the needed alignment and registration between loadable screen holder 200 and storage screen holder 900. Thus, there might be no direct engagement between loadable screen holder 200 and storage screen holder 900 that would prevent relative vertical motion between those two components, and when screens 300 are all in the loadable screen holder 200, there would be no screens that extend across the interface between loadable screen holder 200 and storage screen holder 900 so as to interfere with relative vertical motion between those two components.

[0096] In an embodiment of the invention, there may exist certain geometric relationships between loadable screen holder 200 and storage screen holder 900 and screen 300. Screen 300 may have a screen width WS in the direction of motion followed by screen 300 upon sliding from storage screen holder 900 to loadable screen holder 200. Storage screen holder 900 may have an external dimension, in that same direction, that is SSW. Loadable screen holder 200 may have an external dimension, in that same direction, that is LSW. It is possible that SSW<WS<LSW. What this means is that a screen 300 may be able to fit completely within loadable screen holder 200. This allows loadable screen holder 200 to be removed from seeding tray 1300 after all screens 300 have been slid into it, using a simple vertical motion irrespective of whether storage screen holder 900 is moved at the same time. Also, this relationship provides that prior to the seeding operation, when all of the screens 300 are located in storage screen holder 900, the screens 300 already extend slightly into the wall thickness of loadable screen holder 200. This extending provides that the screen 300 already extends past that interface and past the start of grooves in loadable screen holder 200, which makes it is unlikely for a screen 300 to become stuck at the interface between storage screen holder 900 and loadable screen holder 200. It is possible that when screen 300 is in storage screen holder 900, screen 300 only extends into the wall thickness of loadable screen holder 200 but not any further than the wall thickness, so as to avoid interfering with the motion of pipettes 2020 and the dispensing of fluid from pipettes 2020 in the interior space of loadable screen holder 200.

[0097] This situation can also be described as a situation in which one of the screens 300 can reside in the loadable screen holder 200 without extending outside an external envelope of the loadable screen holder 200, but the screen 300, when residing in the storage screen holder 900, extends outside an external envelope of the storage screen holder 900.

[0098] This relationship can further be described as a relationship such that one of the screens 300 can reside in the loadable screen holder 200 without extending outside an external envelope of the loadable screen holder 200, but the screen 300 when residing in the storage screen holder 900 may extend outside an external envelope of the storage screen holder 900. If the loadable screen holder 200 or the storage screen holder 900 has an exterior surface that is generally planar except for local inwardly-directed features, the envelope can be considered to be the flat plane that contacts those parts of the nearby exterior that are flat.

Auxiliary components related to sterility

[0099] In an embodiment of the invention, there may be provided a storage screen holder 900 and a loadable screen holder 200 assembled to each other, and screens 300 present in the storage screen holder 900, with all of these components being sterile and packaged inside sterile packaging prior to performing a seeding procedure. It is possible that all of these components assembled to each other in a sterile condition and may be enclosed in sterile packaging and shipped as such. Shipping entails the possibility that during shipping the assembly might occupy any orientation in space. It would be desirable that during such a situation, the screens 300 be constrained so that they are not able to slide or shift their position. Accordingly, referring now to Figure 12A, in such an assembly there may be provided a stop 1400 that is suitable to prevent the screens 300 from moving out of the storage screen holder 900, for example during shipping of such an assembly. Stop 1400 may be a flexible or partially flexible piece that touches some portion of screens 300 and some portion of the interior of loadable screen holder 200 in such a way that stop 200 prevents movement of screens 300 when stop 1400 is in place. The stop 1400 may have elasticity and an appropriate shape so that it maintains itself in a position wherein it can prevent the screens 300 from moving, and yet the stop 1400 may be easily be removable by the user prior to a cell seeding process, i.e., prior to sliding any screen 300 from storage screen holder 900 to loadable screen holder 200. The stop 1400 may be a piece of cardboard or other paper-derived material or polymeric material (such as polystyrene) that is folded into an angled shape as illustrated in Figure 12A. The stop 1400 could, as illustrated, be a V-shape having one fold or bend. It could be a truncated V-shape having two folds or bends. It would also be possible for the stop 1400 to be elastically curved or bent in a more general shape. Stop 1400 may be sterile and may be sterilized at the same time as the other packaged components.

[00100] It is also possible to provide a lifting handle suitable to engage with and lift loadable screen holder 200. An example of a lifting handle 1500 is illustrated in Figure 12B in isolation and in Figure 12C in conjunction with loadable screen holder 200. Lifting handle 1500 may be suitable to engage with holes 260 in loadable screen holder 200, and to disengage when desired. Lifting handle 1500 may be deformable between two configurations, so that in a first configuration it may be able to be moved into space near loadable screen holder 200 or in the interior of loadable screen holder 200, and in a second configuration it may engage with loadable screen holder 200. Lifting handle 1500 may be supplied in a sterile condition. Lifting handle 1500 may have a geometry such that the hands of the user who grasps it are maintained some distance away from the actual loadable screen holder 200 and the screens 300 contained therein, so as to help maintain sterility of loadable screen holder 200 and the screens 300. Lifting handle 1500 may be sterile and may be packaged either with the other components or separately.

Pusher and Motion Control System

[00101] There may be provided a pusher 1100 suitable to push screens 300 from a storage position in storage screen holder 900 to a loading position in loadable screen holder 200. Such a pusher is illustrated in Figure 13 A and 13B. Pusher 1100 may be attachable to and detachable from a motion control mechanism that drives and positions it. Pusher 1100 may be a one-time use component that may be supplied in a sterile condition in packaging appropriate to maintain its sterility. Pusher 1100 may be supplied in a sterile condition so as to help avoid contaminating the screens 300, and may be shipped in appropriate sterile packaging.

[00102] As illustrated, pusher 1100 may enter the space of storage screen holder 900 through a side of storage screen holder 900. Storage screen holder 900 may have appropriate opening(s) to allow pusher 1100 to pass through the side of storage screen holder 900 in all of the allowed positions of pusher 1100 so that pusher 1100 may reach screens 300 as needed to touch and move screens 300. Seeding tray 1300 may also have appropriate opening 1310.

[00103] In Figures 13A-13B, the components are exploded for convenience of illustration. It is illustrated that pusher 1100 may attach to the motion control mechanism in a defined location and be retained by magnetic force. One or the other of pusher 1100 and the corresponding motion control mechanism may contain a magnetic component, and the other may contain an appropriate magnetic or metallic component. The interface may include features or dimensionally controlled surfaces to define the location and orientation of the pusher 1100 when it connects to the motion control system. Such features could for example be a post and corresponding recess, or could be flat dimensionally controlled surfaces, or could be other types of dimensionally controlled surfaces. The magnetic features could be either part of dimensionally controlled surfaces or separate from dimensionally controlled surfaces.

[00104] As illustrated in Figures 13A-13B, pusher 1100 may be the component that contacts and pushes a screen 300 and may be horizontally oriented. There may further be provided a support 1110, which may be connected to or be part of a motion control system. Pusher 1100 may have two locating pins 1120 as illustrated, which may cooperate with corresponding locating holes or recesses 1130 in support 1110 to define the location of pusher 1100. A magnet 1140 may be permanently mounted in support 1110. There may further be provided a removable post 1150, some of which may occupy or pass through hole 1160 in pusher 1100. Post 1150 may be made of a material that is attracted by magnet 1140. The interaction of pusher 1110 and related components including magnetically-involved components may be such that if a force or deflection is accidentally exerted or imposed on pusher 1100, the components of the magnetic joint may partially tilt or separate to absorb that force or deflection, and may return to their original position after removal of that force or deflection, as caused by the magnetic attraction. Pusher 1100 may be provided in a sterile condition and may be a one- time-use component.

[00105] Pusher 1100 may be mounted on a motion control system suitable to allow or direct motion of pusher 1100. For example, pusher 1100 may have a degree of freedom of motion in the direction needed to push screens 300 from storage screen holder 900 into loadable screen holder 200. It is possible that such degree of freedom is the only degree of freedom of motion that is provided for pusher 1100. Alternatively, other or additional degrees of freedom of motion could be provided. Pusher 1100 may be driven by a drive system or motion control system 2060 in the appropriate directions of motion. The motion of pusher 1100 may be in the left-right direction as viewed from the front of the system.

[00106] Pusher 1100 can be generally horizontal in its orientation during use and its motion can be horizontal, and its length can be at least the dimension of the screen 300 along the direction of the direction of motion of the screen during sliding from. Pusher 1100 may have a pusher thickness (in the vertical direction) that is less than two times the screen-to-screen spacing distance of screens 300 when the screens 300 are in position in storage screen holder 900 or loadable screen holder 200. However, other geometries and orientations of pusher 1100 are also possible.

[00107] Referring now to Figures 14A-15, there may be provided a motion system suitable to provide desired relative positions of pipettes 2020 and pusher 1100 and loadable screen holder 200 and screen 300. During the course of loading a set of screens 300 in loadable screen holder 200, varying numbers of screens 300 may be present in loadable screen holder 200, and deposition of cells by pipettes 2020 may be performed onto whatever screen 300 is the uppermost screen 300 in loadable screen holder 200 at a given time.

[00108] In an embodiment of the invention, the motion control system may be such that one axis of motion is provided by a device that moves the assembly of screens 300 and screen holders 200, 900, and another axis of motion is provided by a device that moves the pusher 1100, and another axis of motion is provided by a device that moves the pipettes 2020. These three axes of motion may be mutually perpendicular to each other. For example, as illustrated, the driver that moves the assembly of screen holders and screens may move them in the vertical direction. The driver that moves the pusher 1100 may move the pusher 1100 in a horizontal direction, left-right in the perspective viewed in Figure 15. The driver that moves the dispenser or the pipettes 2020 may move them in a horizontal direction, forward-backward in the perspective viewed in Figure 15. As illustrated in Figure 15, the pusher 1100 can be driven in a horizontal direction (left-right in the illustrated view) by horizontal drive 2060. There may be a platform holding the loadable screen holder 200 and the storage screen holder 900 and the reservoir 1000, and the platform can be driven in the vertical direction by vertical drive 2070. The dispenser 2000 can be driven in the horizontal direction (front-back in the illustrated view) by horizontal drive 2080.

[00109] In the system as illustrated in Figures 14A-15, it is not necessary that the vertical elevation of the loadable screen holder 200 be the same during deposition of the cells as it is during sliding of the screen 300 from the storage screen holder 900 to the loadable screen holder 200. As illustrated, the pusher 1100 has a fixed elevation in the vertical direction, while being able to translate in a horizontal direction appropriately to cause the desired sliding of screens 300 (due to its horizontal drive 2060). The motion control system may be operable appropriately to position the seeding tray 1300 and the storage screen holder 900 and the loadable screen holder 200 appropriately to cause the sliding of a desired screen 300, and further may be operable appropriately for cell seeding such that the exposed screen 300 in loadable screen holder 200 is positioned at a desired position or elevation relative to pipettes 2020. Thus, in the system as illustrated, it is unnecessary for the pusher 1100 to have a vertical position adjustment mechanism, and it is unnecessary for the pipettes 2020 to have a vertical position adjustment mechanism. Although it is sufficient to provide, as illustrated, only one vertical motion mechanism, alternatively it is also possible to provide more than one vertical motion mechanism, if desired.

[00110] In addition to what is described, other arrangements are also possible if desired. It would be possible, for example, to provide more degrees of freedom of motion for the screen holder assembly and fewer degrees of freedom of motion for the pusher 1100. Alternatively, it would be possible, for example, to provide more degrees of freedom of motion for the pipettes 2020 and fewer degrees of freedom of motion for the loadable screen holder 200.

[00111] The apparatus may also comprise automatic controls, drive systems, motion control systems, and software appropriate to operate the described components in desired sequences. Stepper motors may be used. Sensors such as encoders may be included as desired. Feedback control or servo systems may be provided.

Dispensers

[00112] Referring now to Figures 14A-15, there is shown a dispenser 2000 suitable to dispense cells for seeding the screens 300. The dispenser 2000 may be suitable to dispense a fluid such as a liquid (which may be a culture medium) containing cells suspended therein. Such apparatus may be similar to pipettes for dispensing biological samples or substances into plates such as conventional microtiter plates (containing 96 wells, 48 wells, or other numbers of wells) that are commonly used for various biological assays, such as for high throughput screening. The dispenser 2000 may comprise an array of pipettes 2020 in a uniformly spaced array, or any other desired arrangement. For clarity of illustration in Figures 14A-15, seven pipettes 2020 are illustrated, but any number of pipettes 2020 could be used. The pipettes 2020 are shown as being identical to each other, but they could be physically different from each other if desired. They could be operated identically to each other, or they could be operated differently from each other if desired. The dispensing action of the pipettes 2020 may be determined by an automation system, which may be computer-controlled. The fluid may be dispensed onto the screen 300 that is uppermost in the stack in loadable screen holder 200 at any given time. An appropriate amount of fluid may be dispensed containing an appropriate number of cells to accomplish the desired seeding. Dispensing may be done so as to avoid causing excess fluid to drip away from screen 300. In Figures 14A-14C, certain components of the overall system are omitted for clarity of illustration. It can be understood that although the term pipette is used illustratively, it would also be possible to use other forms of fluid dispensing including those related to inkjet technology.

[00113] It is further possible to provide a reservoir 1000 of fluid. Reservoir 1000 may contain a sufficient amount of fluid to be deposited on the screens 300 as desired during a seeding process. Such fluid may contain both liquid and cells suspended in the liquid.

[00114] Reservoir 1000 may be mounted on 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 also is raised and lowered. Reservoir 1000 may be positioned such that pipettes 2020 can draw fluid from reservoir 1000 if the tips of the pipettes 2020 are positioned below the surface of the fluid in the reservoir 1000.

[00115] During operation of the system, it is possible to cause an oscillation of the level of fluid in the pipettes 2020 by alternately taking fluid into the pipettes 2020 from reservoir 1000 and then ejecting fluid from pipettes 2020 into reservoir 1000. Such oscillation of the fluid level in the pipettes 2020 can serve the purpose of creating motion in the fluid in the reservoir 1000. This motion may serve to mix the fluid in the reservoir 1000 and counteract any possible tendency of cells suspended in the fluid in the reservoir 1000 to settle out or stratify in the fluid in the reservoir 1000. It can be expected that after several oscillations of the fluid level in the pipettes 2020, the fluid in the reservoir 1000 may have a more uniform concentration of cells than might be the case without such mixing, and the fluid that is finally drawn into the pipettes 2020 for dispensing onto screens 300 may be more representative of the intended concentration of cells than would be the case without such mixing. For example, during mixing, the flowrate either into or out of the pipette can be larger (perhaps several times larger or even an order of magnitude larger) than the flowrate that is dispensed during dispensing of drops onto screens 300. During this oscillatory flow, the flowrate into the pipette 2020 can be different from the flowrate out of the pipette 2020.

[00116] As illustrated, seven pipettes 2020 are illustrated dispensing fluid onto the screens 300, and the pipettes 2020 are arranged in a single row. However, it can be understood that other numbers and arrangements of pipettes 2020 are also possible. Pipettes 2020 can be physically identical to each other or can be different from each other. Pipettes 2020 can be operated identically to each other or differently.

Methods of seeding

[00117] Embodiments of the invention may comprise a method of seeding cells onto a plurality of screens in a screen holder.

[00118] The placement of cells on screens 300 may be controlled by instructions given to the motion control system and to pipettes 2020. It is possible that the density of seeded cells could be uniform over the area of a screen 300 and all the screens 300 could be seeded identically to each other. However, it would also be possible to achieve other distributions of cells if desired. For example, the density of deposited cells could vary as a function of position within a screen 300, if desired. Also, different distributions of seeded cells could be provided on different screens 300, if desired. Any such variation could be achieved by appropriate software or instructions to the system. Even if the system is merely operated so as to provide seeding that is uniformly distributed, such operation can provide assurance that uniform seeding is achieved, with a greater degree of assurance than is possible with certain other cell seeding procedures or techniques.

[00119] An example, a procedure for seeding cells onto a stack of screens 300 in a loadable screen holder 200 may comprise the following steps:

• Load screens 300 into storage screen holder 900. This loading may be done by hand, and may fill all available grooves in storage screen holder 900 with screens 300. At this point in the process, the screens 300 may be dry.

• Align and adjoin storage screen holder 900 and loadable screen holder 200, and register both screen holders or their assembly in position in seeding tray 1300 or in the machine. • Position storage screen holder 900 and pusher 1100, relative to each other, such that pusher 1100 can urge the lowest screen 300 in storage screen holder 900, without urging any other screen 300 in storage screen holder 900.

• Slide the lowest screen 300 from the storage screen holder 900 into the loadable screen holder 200. The pusher 1100 can then be withdrawn to a retracted position.

• Raise the platform holding the reservoir 1000 and storage screen holder 900 and loadable screen holder 200, so that the tips of pipettes 2020 are below the level of fluid in the reservoir 1000. Oscillate fluid level in the pipettes 2020 several times. Lower the platform so as to be able to move the pipettes 2020 out of the vicinity of the reservoir 900.

• Move the pipettes 2020 (moving forward, in the illustrated orientation) so that the pipettes 2020 are above the interior of loadable screen holder 200, and then raise the platform (including loadable screen holder 200 and storage screen holder 900), until the tips of pipettes 2020 are at a desired location relative to the screen 300 that is currently exposed (uppermost) in loadable screen holder 200.

• Dispense fluid containing liquid and cells from pipettes 2020 onto the screen 300 that is currently exposed (uppermost) in loadable screen holder 200.

• Lower the platform so that the tips of pipettes 2020 clear the wall of loadable screen holder 200, and move the array of pipettes 2020 back to reservoir 1000. Raise the platform so that the tips of pipettes 2020 are below the surface of the fluid in the reservoir 1000. Oscillate the fluid level in pipettes 2020 and end when pipettes 2020 are full.

• Reposition storage screen holder 900 and the pusher 1100 so that the pusher 1100 is in position to contact the lowest screen 300 remaining in storage screen holder 900, and urge that screen 300 into loadable screen holder 200.

• Vertically move the platform and horizontally move the array of pipettes 2020 so that pipettes 2020 are in position to deposit fluid onto newly-positioned screen 300.

• Deposit cells onto the newly-positioned screen 300 in loadable screen holder 200.

• Continue these steps in sequence until all screens 300 have been transferred from storage screen holder 900 to loadable screen holder 200 and have been seeded with cells. In order to perform these steps with a particular screen 300, the vertical position of storage screen holder 900 may be adjusted appropriately so that pusher 1100 pushes on a particular screen 300. The vertical position of pusher 1100 relative to storage screen holder 900 can change for each individual screen 300 to be moved.

• As the procedure progresses, storage screen holder 900 will empty of screens 300 starting at its bottom and progressing until it is empty of screens 300, and loadable screen holder 200 will fill with screens 300 starting at its bottom and progressing until it is full of screens 300.

[00120] It can be understood that the vertical elevation of the platform can vary according to which screen 300 is receiving dispensed fluid from pipettes 2020. Also, the vertical elevation of the platform at the time of sliding a screen 300 from the storage screen holder 900 to loadable screen holder 200 does not have to be the same as the vertical elevation of that screen 300 when pipettes 2020 are dispensing fluid onto the screen 300.

[00121] It can be understood that the fluid level in the reservoir 1000 may change during the process. Accordingly, the vertical elevation of the reservoir 1000 at the time of filling the pipettes 2020 or at the time of performing oscillation of fluid in the pipettes 2020 may be chosen appropriately so that the tips of the pipettes 2020 are below the level of the fluid in the reservoir 1000. It is possible to calculate the volume of fluid remaining in reservoir 1000 as a function of steps that have already been performed during the process, and to adjust process parameters accordingly. Such adjustments could involve the relative vertical positioning of the reservoir 1000 and the pipettes 2020 during times when fluid is being taken in to or ejected from pipettes 2020, or the flow parameters of the fluid intake or ejection, or any other parameter of interest.

[00122] It is possible that the pipettes 2020 do not move relative to screens 300 during the actual times when liquid and cells are being dispensed onto screens 300; or, alternatively, if desired, motion and dispensing could occur simultaneously. It would alternatively be possible that the pipettes 2020 or a similar dispenser could move in some predetermined path relative to screen 300 during deposition, such as a raster path. As a still further alternative, it would be possible to deposit a spray of cell-containing liquid from a dispenser, which could be either stationary or moving with respect to the screen. The amount of fluid dispensed from an individual pipette 2020 in one dispensing can be controlled as desired. As illustrated, with only seven pipettes dispensing onto a screen 300 with a fairly large spacing between pipettes 2020, it can be expected that an individual dispensed drop from an individual pipette 2020 will land on screen 300 as a distinct drop separate from what is deposited by a neighboring pipette 2020. However, it also can be expected that the drops deposited on a screen 300 will spread out and merge with each other due to capillary forces and other physical mechanisms. Dispensing as a continuously flowing stream or ribbon would also be possible.

[00123] In an embodiment of the invention, the various pipettes 2020 may be aimed at corresponding target locations within the screen 300 that vary in nature or local geometry from one pipette 2020 to another. For example, one pipette 2020 may be aimed at a location that is a crossing point where a fiber in one layer touches a fiber in an adjacent layer, while another pipette 2020 may be aimed at a location that is not a crossing point. Thus, among various pipettes 2020, there may be some variation or randomness about the local nature of the screen geometry where various drops land on the screen 300. Alternatively, if the spacing between pipettes 2020 were an integer multiple of the repeating dimension of the fibers in the screen 300, it would be possible for all of the pipettes 2020 to aim at screen locations that had the same geometric nature. It is possible that the amount of fluid deposited in one location by a pipette 2020 will be large enough to involve a substantial number of fibers 500, with the result that the details of location of a deposited drop, relative to a fiber or a space between fibers, do not make a significant difference in the depositing and spreading of the fluid.

[00124] In an embodiment of the invention, it has been found that as a result of design trade offs and experiments regarding attempting to provide a desirable amount of open space in which cells can grow, it may be desirable for the inter-fiber spacing to be slightly larger than the diameter of an individual fiber. Neighboring layers can alternate with each other in the direction of their fibers. In such a situation, if the layers that have fibers parallel to fibers in another layer are staggered with respect to each other, that will reduce the amount of open space see-through space and will better help to retain fluid (culture medium and cells) that is deposited onto the screen. It has been found desirable if the maximum dimension of a see- through region is still less than a fiber diameter. This can be achieved as shown in Figures 5E- 5H. This has been found to provide an acceptably small risk of seeding liquid (culture medium and cells) dripping through the screen and failing to seed. A further parameter relevant to seeding is the volume of deposited fluid in comparison to the available free volume of a unit cell. It may be desirable that during seeding, an amount of fluid be dispensed onto the screen such that the dispensed fluid can absorb into or be stored within the screen but not drip out of the screen. For example, if the dispensers are arranged in a rectangular array with the spacing of typical wells in a microtiter plate, which is a spacing of 9 millimeters in a square array, then a unit cell of a screen would be a 9 mm x 9 mm space. In the other dimension, if the screen contains four layers of fibers each having a 150 micron diameter, the overall height of the screen is 0.6 mm. Then the unit volume is 9 mm x 9 mm x 0.6 mm or 48.6 mm^A3. It is found by geometric calculations that if the fiber diameter is 150 microns and the spacing between facing edges of the fibers is 200 microns, then the fraction of space that is occupied by fiber is 34% and the fraction of space that is empty space is 66%. Thus, in the unit cell, the empty space is 66% of 48.6 mm^A3, or 32 mm^A3. It is found experimentally that a good dispensed liquid volume is 15 mm^A3 for that unit cell, while a dispensed liquid of 23 mm^A3 for that unit cell is excessive (resulting in dripping from the screens etc.). These dispensed fluid volumes can be compared to a unit cell empty volume of 32 mm^A3, to give a description that satisfactory deposition occurs at 47% occupancy of the empty volume in a unit cell, but if the occupancy is 72% occupancy of the empty volume in a unit cell, that represents an excessive amount of fluid. So, a suitable range for operation could be 40% to 70% occupancy of the empty volume in a unit cell, or more particularly 45% to 65%.

[00125] In an embodiment of the invention, it is possible to seed cells at a density of approximately 1200 to 5000 cells/cm^A2 of surface area of the screen (referring to the curved surface area of all the individual fibers in the screen. It is believed that a seeding efficiency of approximately 90% can be achieved (i.e., of the cells that are deposited during seeding, approximately 90% of them will remain attached to the scaffold).

[00126] The dispenser may be operated by an automated system and may be programmed. In an embodiment of the invention, the pipettes and other components of the system may be operated so as to deposit or dispense fluid uniformly everywhere in the screen 300. This can provide uniform distribution of liquid and cells from place to place within screen 300, and from one screen to another screen, and can provide repeatability from batch to batch performed by this automated process. This is an improvement over hand pipetting of liquid and cells onto scaffolds.

[00127] After completion of seeding, in order to perform culturing of cells, it is possible to move the seeded scaffold into a bioreactor and to submerge the seeded scaffold gradually in liquid culture medium in a bioreactor. The submerging may be done by raising the liquid level in the region of the loadable screen holder 200. This may be done by operating a pump in the bioreactor system. This may be done sufficiently slowly so as to not dislodge seeded cells. This may be done either continuously or in steps.

[00128] Harvesting, after completion of cell culturing, is a procedure to dissociate cells from the scaffold after culturing. Harvesting can comprise exposing the scaffold to a mechanical vibration while also exposing the cells to a particular chemical environment that promotes detachment of cells. Furthermore, the design of the loadable screen holder 200 is such that flow of culture medium is possible through the open interior of the loadable screen holder 200. Such flow may be in a generally upward direction, passing through the open spaces of the screens 300. This harvesting may be done while screens 300 (as illustrated, 15 such screens) are in place in the loadable screen holder 200. In such a method, cells that become detached may be carried by the flowing medium upward and out of the culture region.

Features related to sterility

[00129] Any of various features may be provided that are useful for maintaining sterility.

[00130] It is possible that loadable screen holder 200 and storage screen holder 900 and screens 300 contained in loadable screen holder 200 can be shipped to the customer in a pre-sterilized condition. These components can be already assembled to each other together with seeding tray 1300, and all of these components may be contained inside appropriate packaging to maintain sterility. It is possible that all of these components may be sterilized together after they have been assembled to or with each other. Sterilization of any component or group of components may be performed by any known sterilization method, including but not limited to gamma ray sterilization, ethylene oxide sterilization and heat sterilization. Components may be appropriately packaged, either before or after the sterilization process, so as to remain sterile.

[00131] It is possible, as discussed herein, that seeding tray 1300 may have gripping handle 1320. Gripping handle 1320 may project outward from the remainder of seeding tray 1300 and may be such that a user can grasp gripping handle 1320. As a result, a user may be able to carry seeding tray 1300 and loadable screen holder 200 and storage screen holder 900 and any screens 300 contained therein, while the user’s hand remains at some distance from those components.

[00132] In view of the possibility of unpredictable orientation of cargo during shipping, it is possible to provide stop 1400 as described herein. Stop 1400 may be discarded thereafter. Stop 1400 may be sterile and may be in included in the assembly when the assembly is sterilized.

[00133] Similarly, it is possible to provide a lifting handle 1500 to aid in lifting and moving loadable screen holder 200 and screens 300 contained therein. Lifting handle 1500 may be provided in a sterile condition, and may be a one-time use component. Lifting handle 1500 may be packaged with the assembly when the assembly is sterilized, or alternatively it could be separately sterilized and packaged.

[00134] Pusher 1100 may also be a one-time use component and may be provided in a sterile condition. Similarly, any other desired auxiliary components can also be provided in a sterile condition.

Further comments

[00135] In still other embodiments of the invention, it would be possible that pusher 1100 could still have motion in a generally horizontal direction as illustrated, but the pusher 1100 itself could have a different orientation such as for example vertical orientation, approaching the loadable screen holder 200 from below.

[00136] In general, any combination of disclosed features, components and methods described herein is possible. Unless otherwise specified, steps of a method can be performed in any order that is physically possible.

[00137] All cited references are incorporated by reference herein.

[00138] Although embodiments have been disclosed, it is not desired to be limited thereby.

Rather the scope of the invention should be determined by the appended claims.

Claims

Claims We claim:

1. A system for creating a stack of screens that are seeded with cells, said system comprising: a plurality of screens;

a storage screen holder suitable to hold said plurality of screens;

a loadable screen holder suitable to hold said plurality of screens;

a pusher suitable to urge individual ones of said screens from said storage screen holder to said loadable screen holder;

a dispenser suitable to dispense liquid containing said cells onto one of said screens, wherein said storage screen holder and said loadable screen holder are located in spaced relation to each other so that one of said screens held in said storage screen holder can be slid in a planar translation motion into said loadable screen holder; and

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

2. The system of claim 1, wherein said loadable screen holder and said storage screen holder contain respective grooves that are collinear or coplanar with each other, said screens being able to slide in said grooves from one of said screen holders to the other of said screen holders.

3. The system of claim 1, wherein one of said screens can reside in said loadable screen holder without extending outside an external envelope of said loadable screen holder, but said screen when residing in said storage screen holder extends outside an external envelope of said storage screen holder.

4. The system of claim 1, wherein said pusher has a thickness in a vertical direction, that is less than two times a center-screen-to-center-screen spacing distance in a vertical direction.

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

6. The system of claim 1, further comprising a seeding tray, wherein said seeding tray and said storage screen holder and said loadable screen holder and said plurality of screens are assembled together to form an assembly, and said assembly is provided in a sterile condition inside a packaging suitable to maintain sterility while said assembly is inside said packaging.

7. The system of claim 1, wherein said motion control system comprises a first drive capable of moving said storage screen holder and said loadable screen holder in a first direction; a second drive capable of moving a pusher suitably to slide one of said screens in a second direction; and a third drive capable of moving said dispenser.

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

9. The system of claim 1, further comprising a reservoir, said reservoir being suitable to hold a fluid, wherein said motion control system comprises a vertical drive that is capable of changing a relative vertical position of said dispenser and said reservoir.

10. The system of claim 1, wherein said screen comprises four layers, each layer comprising fibers that are generally straight and generally parallel to others of said fibers in said layer, said fibers being generally perpendicular to fibers in an adjacent layer of said screen, wherein said fibers in said layers are staggered in a first direction and also are staggered in a second direction that is different from said first direction.

11. The system of claim 1, wherein said screens comprise a plurality of crossed fibers, wherein said fibers have a contact angle with pure water that is less than 50 degrees.

12. A method for creating a stack of screens that are seeded with cells, said method comprising the steps of:

providing the system of claim 1;

operating said system such that a lowest one of said screens in said storage screen holder, being a first-moved screen, is moved into said loadable screen holder, and then fluid is dispensed from said dispenser onto said first-moved screen, followed by moving a next-higher screen from said storage screen holder into said loadable screen holder, followed by dispensing fluid onto said next-higher screen.

13. The method of claim 12, wherein said system further comprises a reservoir holding a quantity of said fluid, and further comprising, prior to said dispensing said fluid from said dispenser, taking some of said fluid into said dispenser and then ejecting at least some of said taken-in fluid back into said reservoir, and then taking some of said fluid into said dispenser.

14. The method of claim 12, wherein a flowrate during said taking in of said fluid into said dispenser is different from a flowrate of said ejecting of said fluid into said reservoir.

15. The method of claim 12,

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

dispensing fluid from said individual one of said dispensers onto said screen, in a vertically downward direction generally perpendicular to said screen, wherein a dispensed volume of said fluid, dispensed by said individual one of said dispensers, fills between 40% and 70% of said open volume of said unit cell of said screen.