Hollow cell sheet-engineering based construct culture

Abstract

Scaffold-free hollow cell constructs are made from cell sheets. A device has an outer membrane and optionally one or more forms and / or anchors surrounded by the membrane. The membrane allows cell attachment followed by detachment of cells and secreted ECM as a cell sheet. In a method, cells are grown into a cell sheet. The cell sheet contracts around one or more forms or anchors. The cell sheet remodels into a hollow cell construct attached to the anchors. When a cell sheet is grown in the device, the forms or anchors are surrounded by the cell sheet. Detaching the cell sheet locates the cell sheet around the forms or anchors. The cell construct can be removed from the forms or anchors and used with or without decellularization, for example in in vitro modelling, regenerative medicine or biorobotics.

Description

HOLLOW CELL SHEET-ENGINEERING BASED CONSTRUCT CULTURERELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, US provisional application 63 / 778,239 filed on March 26, 2025 and US provisional application 63 / 868,085 filed on August 21 , 2025, which are incorporated herein by reference.FIELD

[0002] This specification relates to cell culture devices, methods of growing cell constructs, and to three-dimensional cell constructs, for example hollow cell constructs that are essentially free of exogenous scaffolds or biomaterials made using modified cell sheet engineering techniques.BACKGROUND

[0003] International Publication Number WO 2024 / 243691 A1, Anchored Cell Construct Culture, describes scaffold free cell constructs made from cell sheets. In a method, cells are grown into confluent and ECM-rich layers on a membrane. The layers are detached from the membrane and re-located as a scaffold-free cell sheet around two or more anchors. The cell sheet remodels into a cell construct of a different shape attached to the anchors, for example a fiber. This publication is not citable as prior art in some jurisdictions.INTRODUCTION

[0004] This specification describes a method of making hollow cell constructs, hollow cell constructs, and methods of using hollow cell constructs. The method involves culturing a hollow cell sheet and remodeling the cell sheet on an anchor to produce the construct, wherein the anchor preserves a lumen within the cell sheet. The cell sheet and the construct are typically seamless. The hollow cell constructs may be used, for example, for in vitro modelling or in regenerative medicine, e,g. by transplantation into a body.

[0005] Some embodiments of the method use aspects of the anchored cell sheet engineering approach described in International Publication Number WO 2024 / 243691 A1 or other publications by the inventor. Alternatively, other cell sheet engineering techniques (e.g. growing cell sheets on responsive surfaces) techniques known or subsequently developed may be adapted for use in the method.

[0006] In some embodiments, layers of cells are grown attached to an inside surface of membrane (alternatively called a substrate) that defines a lumen. For example the substrate may circumscribe at least parts of a cylinder or sphere. In some embodiments, a mold for the substrate is made in a negative pattern of the desired substrate shape. Optionally, the mold is water soluble. For example, a mold may be made by 3D printing using water soluble sacrificial filaments (e.g. Polymaker™ PolyDissolve™ PVA filament). Optionally, a washing step with water can be used to soften or eliminate 3D printing patterns on a water soluble mold. A resin, for example a silicone-based resin such as PDMS or another elastomer, is cast in the mold. After the resin is cast and cured, the mold is removed. For example, a water soluble mold may be immersed in water and after a period of time (e.g. a few hours) dissolves and releases the substrate. Optionally, a de-molded silicone (e.g. PDMS) substrate may be treated, for example with a tannic acid solution treatment or a plasma treatment, to improve cell adhesion.

[0007] An interior form, frequently referred to as an anchor herein, is provided inside the membrane. In some embodiments, a mold for a membrane is shaped to also produce the anchor and the anchor is cast in the mold with the membrane, optionally using the same or a different material, optionally with integral runners or other structural elements to position the anchor relative to the membrane. Removing the mold or demolding the membrane releases both the substrate and the anchor. In other embodiments, the anchor is made separately but provided within the mold before casting the membrane (e.g. the mold is made around the anchor or the anchor is inserted into the mold). In other embodiments, an anchor is added to a previously formed membrane, before or after growing layers of cells on the membranes.

[0008] Cells are cultured in layers on the membrane, optionally using methods known in cell sheet engineering, but with steps to distribute cells around an interior surface of the hollow membrane. In some embodiments, cells for a new layer are added to media in the membrane periodically, for example every 2-4 days, to produce layers. For each layer, cells are added in multiple steps separated by a waiting period and re-orientation of the membrane (for example 3 steps for a tube structure or 5 steps for a sphere) to disperse cells over the interior surface of the membrane. In each of these steps, cells are added to media in themembrane and the membrane is held in one position for a period of time (for example 2-4 hours) to allow cells to adhere to a lower portion of the surface of the membrane in that position. Then more cells are added to the media and the membrane is held in a new position to adhere cells to another portion (e.g. a new lower portion) of the surface of the membrane. After a selected number of steps, the cells are cultured to form a substantially confluent layer. The membrane rotation and cell addition steps are repeated to form a subsequent cell layer. After culturing multiple layers of cells on an interior surface of the membrane, the culturing done in a manner that has the cells produce an extra-cellular matrix (ECM), the cell layers detach, or are detached, from the membrane. In some embodiments, the cells may detach from the substrate spontaneously, or detachment of the cell layers from the substrate may be accomplished with an external force or other stimulus, or detachment of the cell layers may be initiated, for example by separating part of the cell sheet from the substrate using rubber tipped tweezers or a scraper, and continue spontaneously. The detached cell sheet has a seamless hollow form, for example a tube, a sphere, or a combination of tubular, spherical or other geometric forms. With continued cell culture, the detached cell sheet contracts inside the substrate and comes into contact with the anchor within the substrate. Further contraction is inhibited by the anchor, which thereby preserves a lumen within the construct. With continued cell culture, the cell sheet remodels on the anchor forming a hollow cell construct, for example a tube or dead-ended chamber. After remodeling, the cell construct may be removed from the anchor.

[0009] In some embodiments, the hollow construct can be perfused with liquid or gas and can withstand some pressure, for example a pressure that would be present in an analogous structure in the body. Applications of tubular structures include modeling or regenerating blood vessels and trachea. Applications of enclosed or chamber-like (e.g. spherical) structures include modeling or regenerating heart, lung aveoli, and stomach organs.

[0010] In some embodiments, constructs are formed with multiple cell types. For example, tubes may have a first layer of smooth muscle cell and a second layer of endothelial cells, to produce for example multi-layer and muli-cell type blood vessels. Multiple cell type sheets can be formed against the membrane and then contract together around the anchor.Alternatively, a cell sheet of one cell type can be contracted around the anchor, and a cell sheet of another type can be cultured and detached from the membrane and then contract around the first cell sheet.

[0011] In some embodiments, the patterns on the anchor can be adjusted to, for example, create villus and crypt-like structures similar to the small intestine in the construct.

[0012] Applications of the hollow constructs can include in vitro modelling, for example for drug testing, drug discovery or other forms of research. In other applications, the hollow constructs may be implanted for in vivo regeneration or other therapies, with or without removal of the cells, and optionally with the addition of therapeutic agents such as cells, genes or drugs.

[0013] In other applications, for example if skeletal or heart muscle cells are used, the tubes and spheres can show contraction and be used as miniature pumps for applications such as biorobotics. These constructs can also be loaded with drugs and when contraction happens they can release these drugs. The contraction can be controlled and induced chemically or electrically.BRIEF DESCRIPTION OF FIGURES

[0014] Figures 1A to 1D are a graphical representation of steps in a process of culturing a hollow tubular and spherical cell constructs, the word “spherical” being used for brevity to refer to a hollow sphere or spherical shell.

[0015] Figure 2A is an isometric view of a device for making a hollow spherical cell construct.

[0016] Figures 2B and 2C and cut-away drawings of membranes with integral anchors for use in the device of Figure 2A wherein the anchor in Figure 2B produces a multi-spherical construct and the anchor in Figure 2C produces a spherical construct.

[0017] Figure 3A is an isometric view of a device for making a hollow tubular cell construct.

[0018] Figure 3B is a cut-away drawing of a membrane with an integral anchor for use in the device of Figure 3A.

[0019] Figures 4 and 5 show photos of tubular and spherical constructs remodeling around anchors 1 and 3 days after being released from a membrane.

[0020] Figure 6 shows a) isometric drawings of tubular and spherical PDMS membranes with integral anchors; b) isometric drawings of molds fabricated by 3D printing with water-soluble PVA filaments used as a sacrificial templates for casting the PDMS membranes of panel a; c) assembled culture devices having the membranes and anchors of panel a with caps and supports added, the supports enabling movement of the devices in the directions of the arrows into different orientations following cell addition to create a full coverage of the surface wherein three rounds of cell addition in the tubular device require three rotations using a triangular support, while five rounds of cell addition in the spherical device require five rotations using a cubic support.

[0021] Figure 7 shows the release of cell sheets from a PDMS membrane after initial scraping resulting in the formation of loose cell sheets, which subsequently recognize the anchors and undergo further remodeling to generate mechanically stable constructs in a single continuous process, wherein the three upper photographs show a tubular membrane and construct and the three lower photographs show as spherical membrane and construct.

[0022] Figure 8 shows a) the effect of anchor shape and size on the geometry of biofabricated hollow constructs, wherein constructs with varying inner diameters can be produced by altering the size of the anchor while using culture devices of consistent dimensions; b) a demonstration of the remodeling capacity of cell sheets to conform to complex anchor geometries, including multi-sphere and tubulo-spherical shapes, even when the 2D culture is conducted on simpler spherical membrane surfaces; c) histological analysis (H&E) of hollow constructs showing modulation of wall thickness, wherein wall thickness can be adjusted by changing the size of the anchor and / or by varying the number of cell layers grown during the 2D culture phase; d) immunohistochemical characterization (CD31) of bilayered constructs formed by sequential culture of skeletal muscle and endothelial cells (3 layers each) in tubular culture devices, resulting in architectures that resemble physiological tissue structures such as blood vessels wherein the arrows indicate the area positive for endothelial marker (CD31 +) in each sample demonstrating that cells can maintain their initial relative positioning even after remodeling.

[0023] Figure 9 shows a) semi-automated image analysis workflow wherein regions of interest (ROI) in whole-slide images (WSI) were manually selected in QuPath (Left), wherein the ROI contour and 1000-pm reference grid were overlaid on the original image and exported as SVG files (Center), and wherein a custom Python script was then used to generate masked ROI images and perform wall thickness measurements (Right); b) Wall thickness analysis of biofabricated hollow tubes wherein on the left side polar plots show wall thickness as a function of perimeter angle for all samples within each condition and on the right side violin plots show thickness distributions (pooled per-degree measurements) with overlaid dots representing per-sample means, wherein increasing number of cell layers and using a smaller anchor size resulted in greater wall thicknesses (n equals 3; statistical comparisons were performed on per-sample mean values; n.s.: not significant; *: p-value < 0.05).

[0024] Figure 10 shows a) a device for growing a bifurcated tubular construct in a first culture orientation, b) the device for growing a bifurcated tubular construct in a second culture orientation and c) anchors for use in culturing a bifurcated tubular construct in the device of panels a and b.

[0025] Figure 11 shows a) a cut-away top view and b) an isometric view of membranes and anchors for the device of Figure 10, panels a and b.

[0026] Figure 12 shows a) side views of a membrane for growing a tubular construct without an anchor (top) and with a discontinuous anchor (bottom) showing a removable shaft in an installed positon and c) top views of one part of the discontinuous anchor (left) and the membrane and discontinuous anchor (right).

[0027] Figure 13 is an isometric view of a membrane with a discontinuous anchor for culturing a spherical construct.

[0028] Figure 14 shows oblique and top view (inset) photographs of a) a tubular construct (left) and b) a spherical construct (right).DETAILED DESCRIPTION

[0029] The terms “cell sheet engineering” and “cell sheet” have been used in the art to refer to the production of a sheet-like collection of cells with preserved cell-to-cell junctions, preferably with an extra-cellular matrix (ECM) produced by the cells. The ECM is initially produced while the cells are attached (e.g. adhered) to a culture surface (optionally called a membrane or substrate), though the ECM may be produced and modified by the cells later. Depending on context, the term “cell sheet” usually also indicates that the collection of cells has been released from the culture surface that was used during the process of forming the cell sheet.

[0030] In a method described herein, cell sheets are grown on a curved substrate, which may provide a lumen. The substrate is three-dimensional in the sense that it is not planar. However, the substrate is two-dimensional in the sense that the cells grow on a surface of the substrate rather than within the substrate. Culture on the curved substrate is therefore distinct from culture in an exogenous three-dimensional (3D) scaffold or bioink, such as a biopolymer or hydrogel, that the cells may be dispersed in. Culturing cells dispersed within an exogenous scaffold can result in cell-to-cell junctions and / or cell-to-scaffold attachments that include the exogenous scaffolding material whereas in cell sheet engineering the cells typically separate entirely from the substrate. The methods described herein may be described as “curved cell sheet engineering” to distinguish then from planar cell sheet engineering and from conventional 3D cell culture.

[0031] Referring to Figure 1, an overview of a method of culturing a hollow cell construct is described in 4 steps, represented by panels A, B, C and D. The method is shown in the context of creating a seamless tubular cell construct and a spherical cell construct. In each case, a hollow substrate 10 is provided with an internal anchor 12. The hollow substrate 10 optionally has a removable cap 14, or alternatively or additionally ports in the substrate 10 or cap 14, for adding or removing materials such as cells and media from the lumen of the substrate as required to culture the cells. In step A, cells 16 are added to a lumen of the substrate 10 and allowed to attach to part of the inside surface of the substrate 10. The substrate is rotated, or otherwise re-oriented, and more cells are added until cells 16substantially cover a selected portion of the surface of the substrate 10. After a period of time, step A may be repeated to provide more layers of cells 16. In step B, the cells are cultured to produce a “pre-sheet” 18. The pre-sheet contains one or more confluent layers of cells with cell-to-cell junctions and an extra-cellular matrix (ECM) produced by the cells. In the case of the tubular substrate 10, the pre-sheet 18 (i.e. the cells adhered onto the membrane) is tubular. In the case of the spherical substrate 10, the pre-sheet 18 is spherical. In step C, the pre-sheet 18 detaches from the substrate 10. In the example shown, detachment is initiating by scraping an edge of the pre-sheet 18 from the substrate 10. The remainder of the pre-sheet 18 detaches spontaneously. In step D, the detached pre-sheet 18 becomes a cell sheet 20. The cells remodel the cell sheet 20, causing the cell sheet 20 to contract until it is restrained by the anchor 12. Culture may continue such that the cell sheet 20 continues to remodel on the anchor 12.

[0032] Figures 2A, 2B and 2C show a membrane 10 for culturing a spherical construct. The membrane 10 is optionally made of PDMS and treated to promote cell attachment. The membrane 10 has a spherical inside surface surrounding a lumen accessible through an opening 24. The cap 14 is removable and, when in place, closes the opening 24 to create an enclosed volume within the membrane 10. An inlet 32 of the cap 14 can be fitted with a filter, connector, valve or other fitting (not shown) and provides a path for material (e.g. cells, oxygen or media) to be added into the membrane 10 or removed from the membrane 10. In the embodiment shown, the cap 14 fits over the opening 24 although in other embodiments the cap 14 may fit into the opening 24. Optionally, a recess 30 provides a place for a clamp or twisted wire (not shown) to compress the cap 14 and the opening 24 together.

[0033] Figures 2C shows an anchor 12 for producing a construct in the shape of a single sphere. Figure 2B shows an anchor 12 for producing a construct in the shape of multiple spheres, for example an alveoli-shaped construct. The anchor 12 is supported by one or more struts 26 which hold the anchor 12 in position relative to the membrane 10. Optionally, the anchor 12 or struts 26 can be reinforced, for example with an embedded metal or rigid plastic pin, which may allow the three struts 26 shown to be replaced with one strut 26. In the example shown the struts 26 extend from the inside of the membrane 10 near the opening 24. Optionally, one or more struts 26 could extend from the cap 14.

[0034] A culture device 34 combines the membrane 10 with a cubical support 22. The flat sides of the support 22 allow the membrane 10 to rest in six orientations on a table. In this example the device can be placed in four positions differing by 90 degrees of rotation around a central longitudinal axis of the membrane parallel with the table. In a fifth position the central longitudinal axis of the membrane is perpendicular to the table. In a sixth position the central longitudinal axis of the membrane is perpendicular to the table and the membrane 10 is inverted in the support 22.

[0035] Figures 3A and 3B show a membrane 10 for making a tubular construct. The membrane 10 is optionally made of PDMS and treated to promote cell attachment. The membrane 10 has a tubular inner surface. One end of the membrane 10 is formed with an opening 24. A removable cap 14 can be fitted to the opening 24 to contain cells and media within the membrane 10. When in place, the cap 14 one end of a lumen defined by the membrane 10 to form an enclosed volume. The cap 14 has an inlet 32, which is optionally fitted with a filter, connector, valve or other fitting (not shown) and provides a path for material (e.g. cells, oxygen or media) to be added into the lumen or removed from inside the lumen.

[0036] The other end of the membrane 10 has an integral end wall 28. The end wall 28 is circular and closes one end of the lumen of the membrane 10. The end wall 28 also supports one end of the anchor 12 to hold the anchor 12 in position within the membrane 10. The shape of the anchor 12 is primarily cylindrical but with optional rings which may help to inhibit longitudinal contraction of a tubular construct. Optionally, the anchor 12 could extend from the cap 14. Optionally, the anchor 12 can be reinforced, for example with an embedded metal or rigid plastic pin.

[0037] A culture device 34 combines the membrane 10 with front and rear supports 22. In the example shown, the outer surface of the membrane 10 is in the shape of a triangular prism with radiused edges. The triangular shape allows the membrane 10 to be held aligned in the supports 22. The supports 22 allow the device 34 to rest on a table in a variety of orientations. In this example, the device 34 can be placed on a table in three positions differing by 120 degrees of rotation around a central longitudinal axis of the device 34. The front support 22 also allows the inlet 32 to be straight and open or bent upwards to close the inlet 32.

[0038] The membranes 10 shown in Figures 2A, 2B, 2C, 3A and 3B are made of PDMS cast in a mold. The master mold is designed to provide a negative pattern of the membrane 10. In some embodiments, the mold is 3D printed using water soluble sacrificial filaments (Polymaker™ PolyDissolve™ PVA Filament). A quick washing step with water is optionally used to soften or eliminate the 3D printing patterns. Once the PDMS substrate is cast and cured, the mold and the cured PDMS are immersed in water. After a few hours the mold dissolves and releases the PDMS substrate.

[0039] In some embodiments, a process may include one or more steps such as preculture, culture initiation, maintenance, culture continuation, cell sheet formation and completion.

[0040] In the preculture step, the membrane is made and prepared to receive cells. The preparations may include, for example, treating the membrane to make the membrane more hydrophilic, coating the membrane to encourage the attachment of a certain type of cell, or sterilizing the membrane.

[0041] The culture initiation step may include assembling a culture system around the membrane, filling the membrane with growth media, and adding cells to seed the device 34. The cells may be, for example, progenitor or stem cells. The cells are added in parts separated by rotating the device to distribute the cells in a layer around the inside of the membrane as will be described further below in relation to the culture continuation step. Optionally, enough cells may be added to over the membrane to about 60-90% confluence. The cells are incubated, for example at 37°C or the physiological temperature of the cell animal. After 2-4 days, during which maintenance steps are performed, the cells will have grown to form a completely confluent first (single cell) layer.

[0042] The maintenance step may be performed daily for as long as cells are within the device 34. The maintenance step can include replacing a growth media with a differentiation media or refreshing a media. In some embodiments, stem cells are differentiated and fusing to adjacent cells with refreshed applications of a differentiation media. For other cell types that adhere (rather than fuse) to each other, the fresh media may be more growth media. The maintenance step may also include rotating the device.

[0043] The culture continuation step may be done at intervals of more than a day, for example every 2 to 4 days. At each of these intervals, cells (typically the same or a similar number of cells as were added in the culture initiation step) are added in multiple doses while the device 34 is held in different orientations. These doses are separated by intervals sufficient to allow the cells to attach to the membrane 10 in each orientation. For example, after cells have been added in one orientation, the device 34 is rested on one side for 2-4 hours to allow the cells to adhere to a portion of the membrane 10. After the resting period, the device 34 is rotated and cells are added to adhere to another portion of the membrane 10. The cells collectively cover most, or optionally all, of the interior surface area of the membrane 10 after cells have been added in all of the orientations (e.g. three orientations for the tubular structures and 5 orientations for the spherical). Cells may expand to completely cover the surface of the membrane 10.

[0044] Each culture continuation step adds a layer of cells. A number of culture continuation steps is selected to produce a selected number of layers, e.g. 1 to 10 layers. The cells attach to each other within and between layers and produce ECM, and optionally fuse together while differentiating. Surprisingly the culture device 34 does not need to be constantly moved during cell culture. With the membrane 10 substantially filled with media, the cells will grow even while hanging from the top of the membrane 10. However, the membrane 10 could be moved periodically or constantly to disperse any effect of gravity on the cells. For example, maintenance steps which continue between culture continuation steps can include rotating the device 34 once or twice a day.

[0045] The cell sheet formation step can commence once cells have grown and optionally differentiated properly and have produced enough ECM to form stable layers. This may be 2-4 days after the last culture continuation step, with continued maintenance steps in between.

[0046] The cell sheet formation step begins with detaching the cell layers, including the ECM, from the membrane 10 to form a loose cell sheet. As cells establish attachments to each other and / or their ECM, their attachment to the membrane 10 weakens. Once the attachment of cells to each other and their ECM becomes stronger than their attachment to the membrane 10, cells and their ECM may detach spontaneously or can be detachedphysically, for example by scraping and / or pulling. In other embodiments, detachment is initiated by scraping part of the cell layers from the membrane 10, for example, using rubber tipped tweezers inserted through the opening 24 of the membrane 10 after removing the cap 14. It is typically not necessary to separate the cell layers from the membrane 10 all the way to the opposite end of the membrane 10 since any remaining attached part of the cell layers will detach as the already detached part of the cell sheet contracts with continued cell culture. It is also possible to make a cell sheet detach by shaking the device 34. Optionally, the device 34 may be inverted such that the weight of the detached part of the cell sheet helps to pull the cell layers from the membrane 10.

[0047] Once at least part of a cell sheet is released from the membrane 10, the device 34 is optionally kept upside down (i.e. with the cap 14 downwards) while the cell sheet remodels. The inverted position encourages the cell sheet to fall around the anchor 12 without wrinkling. The inverted configuration also helps to prevent the cell sheet from falling off of the anchor 12 before it contracts, or collecting on the closed end of the membrane 10 due to gravity.

[0048] When released, a multi-layer cell sheet is formed without using cell sheet stacking methods. The released cell sheet has a thickness of more than one cell. The cell sheet formation step continues for a period of days wherein further contraction of the cell sheet causes it to remodel around the anchor 12. The term “cell construct” is usually used herein to refer to a remodeled form of the initial cell sheet. In some embodiments, the remodeled form is still sheet-like, though it may become thicker than the initial cell sheet. The remodeled form may have other dimensional changes. For example a tubular cell sheet may remodel into a tubular construct with a smaller diameter and shorter length.

[0049] After a period of time in the cell sheet formation step, a cell construct is formed that is strong enough to be extracted in the completion step. Extraction can include removing the cap 14 and cutting the struts 26 or floor 28 to release the anchor 12. The anchor 12 is then removed from within the membrane 10. The cell construct is pulled off of the anchor 12. The cell construct may stretch over part of the anchor 12 while being pulled from the anchor 12.

[0050] If the construct is released from the anchor 12 shortly after separation from the membrane 10, usually sooner than 48 hours after separation, it may continue to shrink and crumple although not as fast or as much as cell sheets that were never supported by an anchor. If the constructs are kept on the anchors 12 for a sufficient time, typically at least 48 hours or more depending on the cell and tissue type, even after taking the constructs off of the anchors 12, they will retain their morphology with minimal further shrinking.

[0051] The precise behavior and the time required to form a stable construct may vary with cell type. For example, if constructs are made out of muscle cells, they can apply more force, as muscle cells do, and can shrink the construct significantly even if they have been hanging on the anchors for a long time. Longer time on the anchors however will result in more ECM production and remodeling which makes the constructs stronger so that detached muscle constructs can resist this contractile force for a period of time. Pressurizing the inside of a muscle contract may prevent shrinkage.

[0052] The resulting seamless hollow constructs are strong enough to withstand biological pressures. For example, they can be perfused with liquid or air, and they can withstand the pressure. In Figure 14, a tubular construct 46 (left side photograph) and a spherical construct 48 (right side photograph) are each attached to a tube 44. The tubular construct 46 is used to convey a fluid flowing into the tubular construct 46 from the tube 44. The spherical construct 48 receives a fluid from the tube 44 and expands and contracts in response the pressure of a fluid in the tube 44. Applications for such constructs include blood vessels and trachea for the tubular structure and heart, lung alveoli, or stomach for the spherical structure.

[0053] In some embodiments, different cell types can be grown separately on top of each other to form a multiple cell type construct. In one method, one or more first layers are grown on the membrane 10 with a first cell type. One or more second layers having a second cell type are grown on top of the first layers. After the first and second layers are detached, a multi-layer cell sheet is formed and remodels around the anchor 12 with one cell type on the inside of the construct and another cell type on the outside to produce a concentrically separated multi-cell type construct. For example, in the tubular membrane 10 a few layers of smooth muscle cells can first be added, followed by one or more layers of endothelial cells.Once the layers are removed from the membrane 12 and form a cell sheet, they shrink around the anchor 12 to produce multi-layer blood vessels.

[0054] In another method, one or more first layers of the first cell type are grown on the membrane 10 and form their corresponding construct around the anchor 12. One or more second layers of a second cell type are then grown on the membrane 10. A cell sheet of the second cell type is later detached and remodels around the construct that is already hanging on the anchor 12. While the second cell sheet is contracting, it also fuses to the previous sheet. This also results in a concentrically separated multi-cell type construct.

[0055] In some embodiments, the curved cell sheet engineering method described herein does not rely on dispersing cells, before or after gelation, in a scaffold such as a hydrogel or bioink. The method also does not rely on exogenous extra-cellular matrix (ECM) materials (such as collagen, fibrin, Matrigel, fibrinogen, thrombin or mixtures of such materials) to provide a material part of the ECM. In contrast, methods described herein are scaffold-free, meaning that cell constructs may be essentially (e.g. 98% or more) or entirely free of exogenous ECM material and / or synthetic or natural biomaterials used as scaffolds. In some embodiments, the initial attachment of the cells to the membrane endures, depending on the cell type, for at least two days, for at least one week, and optionally for about the first 2 to 4 weeks of the process. Essentially all (e.g. 98% more) of the ECM in the resulting cell construct is produced by the cells.

[0056] However, certain cells only adhere to surfaces treated with certain materials. For these cells, the method may include coating the culture surface (with or without hydrophilic treatment) with a material, for example an ECM or ECM-based material (such as collagen, vitronectin, laminin, gelatin or Matrigel™), or a non-ECM material (for example polydopamine or poly-l-lysine). Coating the culture surface typically involves exposing the culture surface to a dilute solution of the ECM or other material for a period of time and then washing the culture surface before adding the cells. The ECM or other material is thereby present as a coating (e.g. individual molecules forming a molecular brush-like structure) rather than a layer and the resulting construct is still considered to be scaffold-free.

[0057] In some embodiments, the membrane 10 surface is patterned. The use of a patterned surface may induce ECM production while the cells are attached to the membrane10. The patterned surface also facilitates growing multiple layers of cells on top of each other before detachment, possibly by creating a 2.5D sense for the cells. Optionally, the patterns may be used to align cells in a particular orientation relative to an anchor 12.

[0058] In some embodiments, 3D printing a mold produces patterns which, if not removed, are replicated in a membrane 10. Alternatively, other materials, for example thermoplastics or metals, may be used to create a mold. Other techniques of creating patterns such as CNC machining or laser engraving may be used to create patterns in a mold. Meso scale patterns can have 100-500 pm spacing between adjacent features. These patterns can have some moderate effect on cell alignment. Micron scale patterns, thinner features spaced 5-50 pm apart, are typically most influential in aligning the cells. Nano scale patterns, features smaller than 1 micometer (< 1 pm), create surface roughnesses that induce cells to secrete higher amounts of ECM components. Patterns with multiple scales of features can be used.

[0059] Other devices and methods of growing cell sheets may alternatively be adapted for use with the devices and methods described herein. For example, cell sheets may be grown on membranes having responsive surfaces such as polystyrene grafted with N-isopropylacrylamide (PIPAAm). In other examples, cells sheets are grown on a membrane and detached using an enzyme to preferentially digest parts of the ECM and the cell sheet to membrane junction. In another example, cell sheets may be grown as described in International Publication Number WO 2022 / 000086 A1, Self-Assembled Cell Sheet Constructs and Methods of Making Thereof.

[0060] A membrane may be non-porous to bulk liquid flow, or at least capable of supporting a liquid media over the cells. However, a membrane made of a bulk material such as silicone may optionally be permeable to oxygen or other gasses. Membranes may be made of various thermoplastics or thermosets. The membranes may be rigid or elastomeric. Membranes can be formed, for example, by machining, molding, casting or extrusion. Some suitable rigid materials are biocompatible epoxy resins, acrylic resins, polyurethane (Pll) resins, and thermoplastic resins. Elastomeric membranes can include, for example, a silicone such as PDMS (e.g. Ecoflex 30-00™ or Sylgard 184™), thermoplastic elastomers based on poly(butylene adipate-co-terephthalate) (PBAT), urethane rubbers formed forexample by reacting a polyol with an isocyanate, and silicones. Other suitable silicone based membrane materials include silicone urethane resins, silicone acrylate resins and silicone epoxy resins.

[0061] Optionally, a membrane may be coated or treated with a stimuli-responsive material, for example a thermally responsive material. Stimuli-responsive materials may transition between a hydrophobic or hydrophilic state based on an environmental factor such as temperature, pH or ionic strength. In some examples, stimuli-responsive materials include a hydrogel or polymer brush that expands or contracts in response to a change in an environmental factor. After a desired number of cell layers are grown on the membrane, a change in the environmental factor is used to cause a change in the environmentally responsive material to cause the cell layers to separate from the membrane or to assist in separating cell layers from the membrane. However, in some examples a stimuli-responsive material is not used.

[0062] One method for making a membrane involves first making a mold. For example a mold may be 3D printed using a filament made from a solvent, e.g. water, soluble filament such as PVA. In the next step a resin suitable for cell culture is cast in the mold. Once the resin cures, the membrane can be removed from the mold, for example by dissolving the mold. Optionally, a permanent or reusable mold may be used with the substrate released, for example, by disassembling a muli-part mold. In some embodiments, a cell culture device is made of multiple materials, for example multiple elastomeric materials or multiple grades of the same elastomeric material. In some embodiments, the device includes a membrane made of a non-elastomeric material and anchors made of an elastomeric material.

[0063] In a device and method described herein, a membrane is provided with an anchor (which may be a multi-part structure) or otherwise associated with an anchor. In some embodiments, the anchor is integral with the membrane. For example, a mold may be configured to produce a membrane with an anchor in a single casting. In some examples, a mold such as a 3D printed mold is made with anchor shaped cavities. The resin is cast in the mold, thereby producing both the membrane and the anchor. Optionally, the membrane and the anchor can be made out of the same resin, different resins, or different ratios of different resins mixed with each other. In some embodiments, a first resin is poured into parts of amold that form an anchor and optionally allowed to gel but not completely set, and a second resin is added to cover the first resin and into the rest of the mold to form the membrane.

[0064] Alternatively, reusable molds or parts of molds may be made, for example by creating a multi-part mold that can be disassembled to release a membrane and / or by combining sacrificial (e.g. water or other solvent soluble, meltable, compacted powder) parts of a mold with non-sacrificial parts of a mold.

[0065] In some embodiments, elastomeric membranes, for example silicone membranes such as PDMS, are treated with an aqueous solution of a polyphenol to provide a more hydrophilic surface. The polyphenol may be plant based, for example tannic acid or lignin. The polyphenol solution can be sterile-filtered using about 0.2 pm syringe filters. The polyphenol may be applied to the membrane without additional cross-linkers or compounding agents. The concentration and duration of treatment are optionally chosen to produce a contact angle of 20-87 degrees. The preferred contact angle may vary with cell type or surface topography of the membrane.

[0066] In order to improve the attachment of tannic acid, lignin or another polyphenol to the surface of the membrane, the membrane can be pretreated with a caustic solution, for example an aqueous sodium hydroxide solution. Alternatively, the membrane may be pretreated with an oxygen, atmospheric air, or carbon dioxide plasma. Optionally, the membrane is not pre-treated with either the caustic solution or plasma.

[0067] After the polyphenol treatment (e.g. with tannic acid and / or lignin), the membranes may be washed to remove residues. For example, the membranes may be washed with deionized water at least twice to remove the residues. Further treatment with sterile solutions of different ECM components such as vitronectin, laminin, or fibronectin can optionally be performed if cells require specific recognition moieties for attachment. Then, the membranes may be used immediately for cell culture or can be stored at room temperature for a time period of a few weeks or in a refrigerator for a longer time period.

[0068] In some embodiments, a hollow cell sheet is produced as an intermediary or precursor product as part of a method of producing a hollow cell construct. The devices andmethods described herein may also be used to produce hollow cell sheets (e.g. cell sheets analogous to the cell sheets produced as intermediary products) as final products.

[0069] Optionally, anchors may be put in place before, during or after growing the cell layers on the membrane. Anchors may protrude from the membrane through the cell layers or be clear of the cell layers prior to detaching the cell layers from the membrane. In some embodiments, the anchor and the membrane are of similar but inverse shapes. For example, part the culture surface of the membrane may be the inside of a tube while the anchor is some or all of the outer surface of a tube or cylinder. In another example, part the culture surface of the membrane may be the inside of a hollow sphere or shell while the anchor is some or all of the outer surface of a solid or hollow sphere or shell. In other embodiments, the membrane and the anchor have different shapes. For example, part the culture surface of the membrane may be the inside of a hollow sphere or shell while the anchor is some or all of the outer surface of a multi-sphere.

[0070] In some embodiments, a seamless hollow cell construct is created after culturing cell layers attached to the inside of a membrane. The membrane may be circular in at least one direction or otherwise forms a continuous closed shape in at least one direction. Optionally, the membrane at least partially surrounds or encloses an anchor. The membrane surface may be made up, for example, of curved, bent or flat sections but very small radius corners in the concave surface are preferably avoided. The membrane may define a lumen and / or be capable of holding a liquid. Alternatively, a membrane may be inserted into a container, for example a dish or tray, to retain liquid in contact with the membrane.

[0071] The membrane surface may curve or bend in one direction, e.g. to form a tube, or in two directions, e.g. to form part of a sphere. The terms tube and sphere will be used for convenience, but the substrate can have other shapes. For example, a tube may include the inner surface of a hollow cone or truncated cone, or the surfaces of a prism (optionally with rounded comers) between its ends. A sphere may be part of any sort of hollow cavity or plenum, for example an egg-shaped cavity or a polyhedron (optionally with rounded corners). Optionally, the membrane may have an irregular shape. In generally, the inner surface of the membrane extends between a first endless line (e.g. a circle) and a secondendless line spaced apart from the first endless line along a central axis that is enclosed or surrounded by the two endless lines and the surface extending between them.

[0072] Different sizes and shapes of anchors can be used to form biologically relevant constructs. For example, a tube with a sphere connected to the end of the tube can be used to create alveoli that is connected to an alveolar duct. In other examples, the patterns on the anchor can be adjusted to create, for example, villus and crypt-like structures in the small intestine.Applications

[0073] In some embodiments, the hollow cell constructs can be used, for example, to produce in vitro models. The construct can be removed from the anchor and moved to an experimental apparatus. Optionally, the construct can remain attached to the anchor, particularly an discontinuous anchor, and moved with the anchor to an experimental apparatus. In another option, the construct may remain attached to the anchor and the membrane may be incorporated into an experimental apparatus.

[0074] In some embodiments, a construct is removed from an anchor and used for implantation. Optionally, a construct may be decellularized before implantation. Optionally, a construct may be decellularized and re-cellularized with a different cell type, or loaded with genes or drugs, before implantation.

[0075] In some embodiments, whether for implantation or in vitro models, various hollow constructs can be fused together or with sheets, spheroids or fibers to construct assemblies (as further discussed below) reflecting organ-level structure and function.

[0076] In other embodiments, if skeletal or heart muscle cells are used the tubes and spheres can show contraction and can be used as miniature pumps for applications such as biorobotics. These constructs can also be loaded with drugs and when contraction happens, they can release these drugs. The contraction can be controlled and induced chemically or electricallyBioassembly

[0077] A device and process for growing cell sheets is described in US Patent 11,718,830, Silicone-Based Membrane Surface Chemistry and Topography Control for Making Self-Assembled Cell Sheets with Cell Alignment and Positioning, fand in International Application No. PCT / CA2023 / 050779, Device and Method for Making Cell Sheets, filed on June 7, 2023. These applications describe, for example, making patterned molds for by way of 3D printing, casting a resin for example of PDMS on the mold, treating the mold for example with a polyphenol to make it hydrophilic, growing a cell sheet on the membrane, and removing the cell sheet from the membrane for example by scraping and / or pulling.

[0078] International Publication Number WO 2024 / 243691 A1, Anchored Cell Construct Culture, describes scaffold free cell constructs made from cell sheets. In a method, cells are grown into a cell sheet on a membrane. The cell sheet is re-located around two or more pillars extending upwards from the membrane through the cell sheet. The cell sheet remodels into a cell construct of a different shape, for example a fiber, attached to the anchors.

[0079] Aspects of membrane fabrication, membrane treatment, cell sheet formation, cell sheet detachment, movement to an anchor, and remodeling as described in these publications may be adapted for use in the methods described herein. Further, the devices and methods described in these publications and herein, optionally in combination with other methods and devices, can be used to make cell constructs in a range of form factors which can be assembled together. Multi-tissue organs can be made by combining multiple constructs in a bioassembly method. Optionally, standardized constructs can be made with different cell types and / or form factors and assembled to form an organ. Since the cell constructs described herein and in the referenced publications are scaffold free, they can easily fuse with each other and form coherent structures. Some of the cell construct building blocks and their uses are described below.

[0080] Tubes (e.g. cylindrical or other hollow tubes) can be used for vessels, ducts or airways. Sheets (e.g. flat, layered or folded sheets) can be used for linings, barriers or membranes. Spheroids (e.g. compact, irregular or packed structures) can be formed by letting anchorless sheets crumple and fold. These structures can be used for glandular orfiltering units. Fibers (e.g. thin, elongated, bundled or striated fibers) can be used for contraction, tension or structural support. Chambers (for example hollow, expandable, spherical or sac-like chambers) can be used for storage or pumping functions. Filters (for example porous, mesh-like or lattice structures) can be formed by decellularizing sheets or by making sheets with a thin layer or layers of cells. Filter structures can be used for selective passage of substances. Networks (for example interconnected, webbed, or reticular structures) can be formed by connecting multiple fibers or hollow tubes. These structures can be used to create communication pathways.

[0081] Example 1 : Formation of hollow tubes with rabbit myoblasts

[0082] In this experiment, rabbit myoblasts are differentiated to skeletal muscle cells and used to form a hollow tube, following these steps:

[0083] A master mold is 3D printed using Original Prusa i3 MK3S+ and sacrificial filaments (Polymaker PolyDissolve PVA Filament). Nozzle size is 0.4 mm and printing speed is 60 mm / s.

[0084] After the mold is 3D printed, it is washed briefly with DI water to soften or eliminate the 3D printing patterns. It is then air dried to avoid damage to the mold’s integrity.

[0085] PDMS SYLGARD 184 is used for making the membrane (alternatively called a substrate or device) with a 10:1 ratio of the base and curing agent. Curing is performed at room temperature overnight to allow all of the trapped bubbles to be extracted. The membrane is generally as shown in Figure 3B.

[0086] After sterilizing the membrane with 70% ethanol, it is treated with 3mL of sterile aqueous solution of tannic acid with a concentration of 50 mg / mL for 72 hours, after which it is washed three times with deionized water to eliminate any traces of tannic acid. It is then autoclaved. Treatment with other cell attachment components such as vitronectin can be done at this stage as well.

[0087] Rabbit myoblasts (Sigma-Aldrich, RB 150-05) were grown up to 80% confluence in 10cm petri dishes in their growth medium (Sigma-Aldrich, RB151-500). They are dissociated using trypsin and 5*105of them are added to the device in the same growth medium. This is repeated two more times while rotating the tubular culture device by 120°between repetitions to disperse the cells within the device, and optionally to cover the entire internal surface of the device with cells. A 2-4 hour resting time is allowed between these steps (i.e. between adding the cells and rotating the device) such that the cells can adhere. Triangular supports as shown in Figure 57 allow the device to rest in three positions. Other numbers of positions could also be used.

[0088] On day 3 of the experiment, medium is switched to the differentiation medium of the cells (Sigma-Aldrich, 151D-250) plus enough L-ascorbate-2-phosphate to achieve a concentration of 100 pg / mL. This media is refreshed every other day until the end of the experiment.

[0089] On days 4, 6, and 8, the same number of cells (5x105) in similar stepwise manner are added into the membrane directly to the differentiation medium. The newly added cells adhere on top of the previous layers of cells and will differentiate to skeletal muscle cells.

[0090] On day 18, the cells and their ECM were scraped off of the tubular walls of the membrane as a seamless tubular sheet using rubber tipped straight tweezers.

[0091] As shown in the upper part of Figure 4, on Day 1 after scraping the detached cell sheet begins to contract. Optionally, a cap can be placed on the membrane so that it can be inverted as shown in Figure 5. When inverted, the cell sheet can hang temporarily from the closed end of the membrane (some cells may adhere temporarily to the closed end of the membrane) which may reduce contraction of the construct in length. On day 3, the tubular sheet has partially remodeled against the central anchor and further contraction in length is reduced. After sufficient remodeling, the tubular can be removed from the central shaft as a seamless tube, optionally after cutting the central shaft out of the device. The unanchored construct is shown in the two upper right-hand photos of Figure 4.

[0092] Example 2: Formation of hollow spheres with rabbit myoblasts

[0093] The process of culturing a spherical construct is generally the same as in the example above but the membrane has curvature in two directions (rather than 1) and forms a rounded chamber, in this case a spherical chamber. The membrane is held in a support with 5 sides as shown in Figure 5. On days when cells are added, the cells are adding withthe device in the 5 available positions to disperse cells around the inner surface of the membrane. Optionally, other numbers of positions could be used. The cells achieve full coverage of the internal membrane surface in each layer either as they are deposited or after some growth.

[0094] After hollow spherical cell layers form on the inside of the membrane, rubber tipped bent tweezers are used to detach the cell layers from at least a portion of the membrane. The remaining parts of the cell layers may detach spontaneously. As shown in the lower part of Figure 4, on Day 1 after scraping the detached hollow cell sheet begins to contract. Optionally, a cap can be placed on the membrane so that it can be inverted relative to the position shown in Figure 4, as shown in Figure 5. When inverted, the cell sheet can drop over the central anchor. On day 3, the hollow sheet has partially remodeled against the central anchor. After sufficient remodeling, the central anchor can be cut out of the device as shown in the two lower right-hand photos of Figure 4. The hollow cell sheet can later be pulled off of the central anchor. The opening of the hollow spherical construct expands while being pulled over the anchor but then returns to its previous size. Optionally, the anchor may be hollow or in the form of a cage-like structure (similar to the anchor 12 of Figure 13) such that the anchor can be collapsed to make it easier to remove the hollow spherical construct.

[0095] Example 3: Excerpts from The Tissue Engineering Grail: Seamless Biofabrication of Scaffold-fee Hollow Constructs; Alireza Shahin-Shamsabadi and John Cappuccitti, bioRxiv preprint doi.org / 10.1101 / 2025.11.04.685834, posted Novembers, 2025.

[0096] Scaffold-free tissue engineering enables the construction of biomimetic tissues and organs by preserving cell-cell and cell-matrix interactions while avoiding exogenous scaffolds and biomaterials. Yet current approaches are limited to thin sheets or simple spheroids and often lack cellular maturity and organized extracellular matrix (ECM). Here, Anchored Cell Sheet Engineering, a concept that previously introduced anchors to guide the remodeling of cell sheets into more mature fibers or sheets, is extended to achieve seamless, single-step biofabrication of scaffold-free hollow tubular and spherical constructs for sustained biological and mechanical functions under physiological conditions. Using custom culture devices with curved geometries for two-dimensional (2D) culture, continuous confluent cell-ECM layers were formed that were then delaminated and guided bystrategically positioned central cores with different shapes and sizes to undergo tension-mediated remodeling into mechanically stable hollow structures. This approach allows modulation of wall thickness, supports multi-layered architectures, and yields constructs capable of withstanding fluid flow. By expanding scaffold-free biofabrication beyond sheets and fibers to robust hollow geometries, this work establishes a versatile set of physiologically relevant building blocks for scalable bottom-up assembly of complex, multi-tissue organ-like constructs within a bioassembloid framework.

[0097] In this study, the Anchored Cell Sheet Engineering concept is extended to overcome this critical barrier, introducing a method for the single-step fabrication of scaffold-free hollow tubular and spherical constructs. By adapting cell culture to custom devices with curved surfaces, confluent cell-ECM layers are formed and guided to delaminate and remodel around central cores, creating robust, mechanically stable hollow tissues. The platform allows modulation of wall thickness, supports multi-layered architectures through sequential seeding of distinct cell types, and produces constructs capable of maintaining stability under fluid flow. This advancement expands the palette of scaffold-free biofabrication beyond fibers and sheets, establishing hollow structures as standardized building blocks within a bioassembloid framework for assembling complex, multi-tissue organ-like systems for regenerative medicine applications. The method is shown graphically in Figure 4.Device Fabrication

[0098] Culture devices were fabricated using polydimethylsiloxane (PDMS; Sylgard 184) cast onto master molds containing negative designs of the intended culture geometry. Master molds were fabricated using fused deposition modeling (FDM) 3D printing with water-soluble polyvinyl alcohol filament (Polymaker PolyDissolve S1 PVA). Following PDMS curing at room temperature overnight, master molds were dissolved by thorough washing with deionized water to yield the final cylindrical or spherical PDMS-based culture devices. The cylindrical devices featured an internal diameter of 14 mm and height of 45 mm, while spherical devices had an internal diameter of 30 mm. Central anchoring cores of varying dimensions were integrated into each device design (4 and 6 mm shafts and 10 and 14 mm spheres for cylindrical and spherical devices, respectively) to enable tunable construct geometries. Culture device caps were fabricated from another silicone-based resin(Ecoflex™ 00-30) and incorporated inlet tubing designed to accommodate 18-gauge needles for cell seeding and medium exchange. Sealing wires were included to secure connections between caps and culture devices. External supports made with polylactic acid (PLA) fabricated by 3D printing enabled manual rotation of culture devices during cell seeding procedures.Cell Culture and Cell Sheet Engineering

[0099] Culture devices were prepared for cell seeding by treating internal surfaces with aqueous tannic acid solution (Sigma-Aldrich, 403040, 50 mg / mL) for three days to enhance PDMS surface hydrophilicity. Following extensive washing with deionized water and autoclaving for sterilization, surfaces were treated with vitronectin (Gibco™, A31804, Recombinant Human Protein, Truncated, 10 pg / mL) in PBS for 1 hour at room temperature to promote cell attachment. Primary skeletal muscle myoblasts (SkMb, Sigma Aldrich, RB150) were cultured in their growth medium (Sigma Aldrich, RB151). Cell culture was initiated on day 1 by introducing 5x105cells along with growth medium to fill culture chambers (5 mL for cylindrical devices, 12 mL for spherical devices). Cell seeding was repeated every two hours with device rotation (two additional rotations for cylindrical, four for spherical devices) to ensure uniform cell distribution across internal surfaces. On day 3, growth medium was replaced with differentiation medium (Sigma Aldrich, 151D) to induce myotube formation. This medium was refreshed daily thereafter. Additional cell seeding rounds were performed on days 4, 7, and 10 using identical procedures. On day 12, devices were uncapped and cell-ECM layers were gently detached from culture surfaces using 1 mL pipette tips. Devices were recapped and incubated overnight to facilitate cell sheet remodeling and anchoring onto central cores. Cultures were maintained until day 18 to enable completion of remodeling and formation of mechanically stable constructs capable of detachment from anchoring cores.Histological and Immunohistochemical Analysis

[0100] Constructs were fixed in 2 wtA / % paraformaldehyde (Sigma Aldrich, 158127) in PBS for 10 minutes at room temperature, followed by dehydration through graded ethanol series and embedding in paraffin. Serial sections (5 pm thick) were cut using a microtome and mounted on glass slides. Hematoxylin and eosin (H&E) staining was performedaccording to standard protocols for general morphological assessment and wall thickness measurements. For immunohistochemistry (IHC) targeting CD31, tissue sections were initially deparaffinized with xylene and rehydrated through a graded ethanol series of decreasing concentrations. Antigen retrieval was carried out using citrate buffer (pH 6.0) in a pressure cooker for 20 minutes, followed by incubation with 0.3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were then blocked with 10% donkey serum in PBS for 1 hour at room temperature. Primary antibody (Abeam, ab182981) incubation was conducted overnight at 4°C in a humidified chamber. After washing with PBS, the sections were incubated with a peroxidase-conjugated donkey antimouse secondary antibody (Jackson ImmunoResearch, 715-035-150). Hematoxylin was used for counterstaining, after which the sections were dehydrated, cleared, and mounted using a permanent mounting medium. All stained slides were scanned digitally at 40* magnification using a Leica Aperio AT2 scanner (Leica Biosystems, Buffalo Grove, IL) and the results were saved as whole-slide images (WSI).Image Analysis and Quantification

[0101] WSIs were opened in QuPath, where the hollow tubes’ cross-sections were defined as regions of interest (ROI) by manually drawing a closed contour. For each condition, three biological samples were analyzed, with two histological slides taken from different locations of each sample. A reference grid with 1000-pm spacing was overlaid to standardize spatial measurements. For each sample, the underlying image, the contour, and the reference grid were exported together as an SVG file to preserve vector geometry. Analysis proceeded using a custom Python script that isolated the ROI and converted it into a binary annular mask, which, when applied to the base image, yielded a ring-only ROI. Wall thickness was quantified using a normal-based approach. Inner and outer boundaries were extracted from the cleaned mask, and the inner boundary was resampled uniformly by arc length. At each sample point, the local outward unit normal was computed from the boundary tangent, and thickness was measured by casting a short ray along this normal. Distances were converted to micrometers using the grid-derived scale. Results were visualized as violin plots of the pooled thickness distribution for each condition, and as a combined polar overlay of all samples. For statistical analysis, mean wall thicknesses from each sample werecompared across conditions. One-way ANOVA was performed to detect overall differences among groups, followed by Tukey’s honestly significant difference (HSD) post-hoc tests for all pairwise comparisons. Adjusted p-values from Tukey’s test were used to determine statistical significance, with a threshold of p-value smaller than 0.05.Discussion

[0102] The culture devices used in this study consisted of hollow cylinders or spheres, each containing a central shaft or sphere, respectively, to serve as an anchoring core (Figure 6a). These devices were fabricated by casting polydimethylsiloxane (PDMS) onto sacrificial master molds 40 with negative designs of the intended culture devices that were 3D printed using a water-soluble polyvinyl alcohol filament (Figure 6b). Once the PDMS cured, the molds were dissolved with deionized water to yield the final culture devices. The complete culture system included a silicone cap with a tubing inlet for cell seeding and media exchange, a sealing wire to secure the cap in place, and a 3D printed external support to allow for manual rotation, essential for ensuring uniform cell coverage on all internal surfaces of the device (Figure 6c )^

[0103] Before cell seeding, the devices were pretreated with tannic acid to adjust hydrophilicity and promote cell adhesion. This was followed by washing, autoclaving for sterility, and coating with vitronectin for long-term cell attachment. The cell culture process began on day one with the introduction of 5x105primary skeletal muscle myoblasts (SkMb) in growth medium. For uniform and complete cell distribution, the devices were rotated, and the seeding process was repeated every two hours (a total of 3 and 5 times for cylindrical and spherical culture devices). On day three, the growth medium was replaced with a differentiation medium to induce myotube formation, and this medium was refreshed daily. Additional rounds of cell seeding were performed on days 4, 7, and 10. The key cell sheet engineering step was initiated on day 12 when the cell and ECM layers were gently detached from the device walls using a 1mL pipette tips and allowed to remodel overnight around the central anchoring cores (Figure 7). After a total of 18 days in culture, the process yielded robust constructs that could be extracted from the cores and exhibited sufficient mechanical integrity to withstand gas and air flow.

[0104] The versatility of this biofabrication platform was demonstrated by creating hollow constructs of varying shapes and sizes simply by modifying the central core’s geometry (Figures 8a and 8b). While the outer dimensions of the culture devices remained consistent, changing the core size allowed for the fabrication of constructs with different inner diameters (Figure 8a). The cell sheets also displayed a remarkable capacity to remodel around more complex core shapes, such as multi-sphere and tubulo-spherical configurations, even when cultured in a simple spherical device (Figure 8b). The wall thickness of the constructs was measured using a semi-automated workflow that involved manual detection of sample areas in QuPath followed by image analysis using a custom Python script. (Figures 8c and 9). Quantifying H&E-stained images of the hollow tubes confirmed that wall thickness was tunable. For example, using a smaller central core (4mm ID vs. 6mm ID) in the cylindrical devices increased the wall thickness from 78.35±18.30 to 176.16±59.63 pm. Wall thickness could also be adjusted by changing the number of cell layers. Increasing the number of cell layers from four to six resulted in a wall thickness increase from 78.35±18.30 to 159.36±34.04 pm for a large core. Finally, to mimic the more complex native tissue architectures, constructs with multiple cell types were biofabricated. Sequentially layering three muscle cell layers followed by three endothelial cell layers produced a bilayered structure that was preserved during the cell sheet formation and remodelling. Distribution of the endothelial cells within these bilayred constructs that resembles the microarchitecture of blood vessels was confirmed by immunohistochemistry for the endothelial marker CD31 (Figure 8d).

[0105] The results presented here show that planar 2D surfaces can be replaced with curved ones to guide cells into self-organizing into complex, seamless 3D architectures without requiring external scaffolds or manual assembly steps (Figures 6 and 7).

[0106] The method introduced here enables cells to deposit and organize their native ECM, resulting in superior biological relevance and mechanical properties that naturally match tissue requirements. The seamless nature of these constructs represents an alternative or advancement over cell sheet wrapping methods since wrapped structures could potentially exhibit mechanical failure at fusion interfaces under physiological pressures. The single-step process introduced here eliminates these weak points and provides in someembodiments the ability to withstand sustained gas and liquid flow without structural compromise. The tension-mediated remodeling driven by strategic anchor placement may mimic developmental morphogenetic processes where mechanical forces guide tissue organization. Without intending to ne limited by theory, it is possible that as the cell sheet compacts around the anchor, cell-generated tension activates mechanosensitive signaling pathways. This likely involves the nuclear translocation of transcriptional co-activators like YAP and TAZ, which can convert mechanical cues into biological responses. This could increase production and alignment of ECM components, leading to the tissue maturity and mechanical stability observed in the constructs.

[0107] The ability to include multiple cell types and tune wall thickness (Figures 7 and 8) provides control over the biofabrication of highly in vivo-like and physiologically relevant constructs. Complex core geometries (Figure 6), including multi-sphere and tubulo-sphere configurations, may be produced. This approach also enables fabrication of millimeter to centimeter-scale hollow constructs that could be used, for example, as functional tissue units or conduits for larger organ systems.

[0108] The Anchored Cell Sheet Engineering platform now offers a set of standardized building blocks encompassing sheets, fibers, spheroids, hollow tubes, and hollow spheres that establishes a comprehensive toolkit for bottom-up organ assembly following modular tissue engineering techniques such as bioassembloids. This modular approach contrasts sharply with monolithic organ engineering strategies that attempt to recreate entire organs in single steps. The standardized nature of these building blocks enables quality control at the component level before assembly. The natural fusion capacity of these scaffold-free tissue units provides a clear path for assembling these building blocks into larger structures.

[0109] By replacing planar substrates with custom curved geometries and using centrally placed cores as guiding anchors, cells form confluent inner linings that remodel via controlled delamination into mechanically robust, biologically relevant 3D architectures. Dimensions and shapes are tunable through core design, and multiple cell types can be layered to produce biomimetic, multi-layer structures with native ECM deposition and anisotropic alignment. Together with sheets, fibers, and spheroids, these hollow formsconstitute standardized scaffold-free building blocks that underpin the bioassembloid concept for bottom-up assembly of larger tissues and, ultimately, organ-scale constructs.

[0110] Example 4: Bifurcated hollow structures

[0111] Referring to Figures 10 and 11 , one application of hollow tubes is for replacing damaged blood vessels. Bifurcations are commonly observed in vivo for blood vessels and the ability to form such constructs is useful for covering a range of in vivo needs or a range of in vitro models. To create a bifurcated construct, a bifurcated membrane 10 is provided with a bifurcated anchor 12.

[0112] The bifurcated anchor 12 is harder to extract from a cell construct than a tubular anchor. In order to facilitate removing the bifurcated anchor 12, or other anchors with complex shapes, a porous anchor 12 may be used. The porous anchor 22 may be made separately and then inserted into the mold for the membrane 10. The membrane 10 is cast around the anchor 12 and holds the ends of the anchor 12. Alternatively, the membrane 10 may be formed, optionally with indentations of other features to receive the ends of the anchor 12, and a separately formed anchor 12 can be inserted into the membrane 10.

[0113] In Figure 10, panel c, a porous anchor 12 is shown on the left side of the photograph next to a non-porous silicone anchor 12 on the right side of the photograph. A porous anchor 12 can be made with any PDMS or other silicone including various Ecoflex™ products or Sylgard 184™. To make the porous anchor 12, a mold for the anchor 12 is 3D printed with an infill pattern that creates interconnected pores such that a continuous material will remain when the infill pattern is removed. One suitable infill pattern is a Gyroid pattern. The selected infill density determines the stiffness of the anchor 12. The mold is printed with a water-soluble filament and an open top such that the silicone resin can be cast inside of the mold. After the silicone is cured, the printed mold can be dissolved away. Gyroid infill can be used in a range of about 10-60%, with the infill volume selected to control the porosity, pore size, and flexibility of the anchor. If the mold is printed with 10-15% infill, the anchor will have 85-90% resin by volume. Such an anchor will have mechanical properties similar to a solid anchor but will compress slightly when a construct is removed. A mold printed with 50-60% infill will produce an anchor with 40-50% resin by volume. Such an insert provides sufficient support for cell construct remodeling but is flexible and easily compressed to allowfor the anchor to be pulled out of more difficult constructs such as a bifurcated tube. A mold printed with an infill volume of more than 60% typically has pores that are too small to be filled with a silicone resin when a Gyroid infill is used, but might be usable with a different infill pattern or with a less viscous resin and enable even more flexible and compressible anchors to be formed.

[0114] In the example shown in panels a and b of Figure 10, the support 22 outside of the membrane 10 can be rotated through four positions to provide full coverage of cells on the required inside surfaces of the membrane 10 during cell culture. The four positons are the two positions shown in panels a and b of Figure 10, and the inverse of those two positions. The cells do not need a completely horizontal surface to allow for full coverage of the membrane 10. Even with each arm of the membrane 10 sitting with a small angle (as in pane b), the cells can still attach to inside surfaces of the membranes 10 to provide a uniform coverage.

[0115] Example 5: In vitro Modeling on a discontinuous anchor

[0116] In order to use the hollow structures for in vitro modeling, the formed constructs typically need to remain anchored, at least at their openings, preferably while allowing simultaneous access to both inner and outer regions of the hollow constructs. In some embodiments, a tube or other structure is inserted into one or more openings of a construct that has been removed from its culture device to provide such continuous anchoring and access to the lumen of the construct. Alternatively, to avoid removing the construct from its culture device, a discontinuous anchor may be provided within the membrane.

[0117] Referring to Figure 12, in an embodiment for a tubular construct, the anchor 12 has two separate parts, one near each end of the membrane 10. When culturing tubular constructs with a high aspect ratio, there is a risk that part of the construct between the two parts of the anchor 12 could collapse before the construct has fully stabilized. To prevent such collapse, in the initial phases of construct remodeling, a shaft 42 can be inserted between the two parts of the anchor 12. The shaft 42 may also help to provide more uniformity to the construct. After a period of remodeling, the shaft 42 can be removed. Tubes extending from outside of the membrane 10 to the permanent parts of the anchor 12 allow access to the lumen of the hollow construct while it remains on the anchor 12.

[0118] In the example shown, the permeant parts of the anchor 12 are made separately and the membrane 10 is cast around the anchor 13. In this way, the anchor 12 can be made of a hard plastic, which can have features designed for connection later to laboratory equipment such as surgical tubing or a syringe. Optionally, the permanent parts of the anchor 12 can be made with a composite of resin mixed with conductive particles to make electrical stimulation of the tubular construct possible.

[0119] Referring to Figure 13, a similar concept, wherein the cell construct is retained on a discontinuous anchor 12, is used to make a spherical construct. The discontinuous anchor 12 permits access to both the inside and outside of a construct cultured on the anchor 12 for in vitro modeling or testing purposes. In various applications, different treatments (media formulation, drugs, etc.) may be examined by flowing different solutions inside and / or outside of the construct through ports in the membrane 10 or a cap 14 (not shown).

[0120] Optionally, a removable anchor part can be provided inside of the anchor 12 shown in Figure 13. The removable part could be provided by, for example, an inflatable balloon or a porous and / or flexible part as described above for the bifurcated tubular construct. After the construct is sufficiently stable, the removable part can be removed from inside of the discontinuous anchor 12. However, the hollow spherical construct is at low risk of collapsing since one ring of the anchor 12 is larger than the other 12. Accordingly, in some embodiments a removable part for a discontinuous anchor 12 is not required. Optionally, a construct supported from the discontinuous anchor 12 may be filled with a fluid at a higher pressure than fluid outside the construct during remodeling to inhibit the construct from shrinking tightly against the discontinuous anchor 12.

[0121] The following documents, including the supporting information described in them, are incorporated herein by reference: (1) US Patent Application Publication Number US 2025 / 0229001 Al, published July 17, 2025, Anchored Cell Construct Culture; (2) International Publication Number WO 2023 / 240336 A1, Device and Method for Making Cell Sheets, published December 21, 2023; (3) International Publication Number WO 2024 / 243691 A1, Anchored Cell Construct Culture, published December 5, 2024; (4) The Tissue Engineering Grail: Seamless Biofabrication of Scaffold-fee Hollow Constructs; Alireza Shahin-Shamsabadi and John Cappuccitti, bioRxiv preprintdoi.org / 10.1101 / 2025.11.04.685834, posted November s, 2025; (5) Shahin-Shamsabadi, A. and J. Cappuccitti, Anchored Cell Sheet Engineering: A Novel Scaffold-Free Platform for in vitro Modeling, Advanced Functional Materials, 2024 34(13): p. 2308552; (6) Shahin-Shamsabadi, A. and J. Cappuccitti, In Vivo-Like Scaffold-Free 3D In Vitro Models of Muscular Dystrophies: The Case for Anchored Cell Sheet Engineering in Personalized Medicine, Advanced Healthcare Materials, 2024. 34(13): p. 2404465; (7) Shahin-Shamsabadi, A. and J. Cappuccitti, Proteomics and machine learning: Leveraging domain knowledge for feature selection in an skeletal muscle tissue meta-analysis, Helion 10 (2024) e40772; and, (8) Alireza Shahin-Shamsabadi, John Cappuccitti, Muscle-specific acellular ECM fibers made with anchored cell sheet engineering support regeneration in rat models of volumetric muscle loss, Acta Biomaterialia, doi.org / 10.1016 / j.actbio.2025.05.024. In the event of any inconsistency between this specification or its figures and an incorporated document, the provisions of this specification and its figures will supersede the inconsistent statement of the incorporated document.

[0122] The examples provided herein are intended to enhance and further enable the disclosure and not to limit the invention. Other embodiments of the invention may be made or used within the scope of the invention, which is defined by the following claims.

Claims

1. CLAIMS:l / We claim:

1. A method for producing a cell construct comprising the steps of,growing a hollow cell sheet;locating the hollow cell sheet around one or more forms or anchors; and, culturing the hollow cell sheet, whereby the hollow cell sheet remodels to form a hollow cell construct.

2. The method of claim 1 comprising,growing cells in one or more layers attached to the inside of a hollow membrane; and, releasing at least a portion of the one or more layers from the hollow membrane, thereby forming the hollow cell sheet.

3. The method of claim 2 wherein the one or more forms or anchors are located within the hollow membrane and wherein an inner surface of the one or more layers surrounds the one or more forms or anchors.

4. The method of claim 2 or 3 wherein the one or more layers of cells are grown attached to the membrane for at least two days, for at least one week, or for at least two weeks, and produce ECM while attached to the membrane.

5. The method of any of claims 1 to 4 wherein the membrane is treated to permit cell attachment and optionally grooved.

6. The method of any of claims 1 to 5 wherein the cell sheet has multiple layers.

7. The method of any of claims 1 to 6 performed essentially without adding an exogenous ECM material, hydrogel scaffold or bioink.

8. The method of any of claims 1 to 7 wherein the hollow cell construct is a tube or chamber.

9. The method of any of claims 1 to 8 wherein the hollow cell construct comprises a combination of multiple forms, for example multiple tubes such as a bifurcated tube, multiple spheres, or a tube and a sphere.

10. The method of any of claims 1 to 9 wherein releasing the one or more layers comprises scraping edges of the one or more layers away from the membrane, optionally wherein a remainder of the one or more layers spontaneously detaches from the membrane, or activating a stimuli-responsive surface of the membrane.

11. The method of any of claims 1 to 10 comprising decellularizing the construct.

12. The method of any of claims 1 to 11 comprising growing a second type of cells in the construct.

13. The method of any claims 1 to 12 comprising removing the hollow cell construct from the anchor.

14. The method of any of claims 1 to 12 comprising in vitro testing of the hollow cell construct on the anchor.

15. The method of claim 14 wherein the anchor is a discontinuous anchor or includes a permanent anchor and a removable anchor, wherein the removable anchor is removed before the in vitro testing.

16. A seamless hollow cell construct consisting essentially of cells and ECM produced by the cells.

17. The seamless hollow cell construct of claim 16 comprising two cell types in a radially separated configuration.

18. The seamless hollow cell construct of claim 16 or 17 in the form of a tube, sphere, bifurcated tube, multi-sphere or tubulo-sphere.

19. The seamless hollow cell construct of any of claims 16 to 18 that is essentially free of exogenous biomaterials.

20. The seamless hollow cell structure of any of claims 16 to 19 attached to a discontinuous anchor.

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