Monolayer cell patch in an extracellular matrix scaffold

Abstract

This invention provides a method for encapsulating cells in microtissue. [Solution] A method for encapsulating cell microtissues includes coating a tissue scaffold stamp with an extracellular matrix compound. The method includes placing the tissue scaffold stamp on a thermoresponsive substrate and seeding a cell culture onto the tissue scaffold stamp. The cell culture forms cell patches that bind to the extracellular matrix compound. A monolayer boundary on the tissue scaffold stamp maintains expression for intercellular junctions, where the monolayer intercellular junctions are configured to express tension. The method includes removing the thermoresponsive substrate. The method includes folding the microtissue structure by suspending the microtissue in a solvent. The folded microtissue structure is recovered from the solvent and administered to a living organism.

Description

[Technical Field]

[0001] Claim of priority This application claims priority under 119(e) of U.S. Patent Application No. 62 / 973,695, filed on 18 October 2019, which is incorporated herein by reference in its entirety.

[0002] Government rights This invention was made possible with government support granted by the National Institutes of Health (NIH) under authorization number 1RO1EY024642-01A1. The Federal Government has certain rights in this invention.

[0003] This disclosure relates to the formation of microtissues and the application of microtissues to living organisms. [Background technology]

[0004] Administering cells to patients has become a promising therapy for many diseases. However, cells delivered in single-cell suspensions often die immediately after injection into the repair site, and it is unclear whether the single cells adequately repair organs and tissues composed of millions of damaged or incomplete cells. Current methods of cell injection therapy involve the use of enzymatic release of cells into single-cell suspensions, which alters the cellular structure and phenotype. In addition, injected single cells often die immediately after injection due to a combination of stress resulting from the physical injection process and insufficient binding to the desired tissue. [Overview of the project]

[0005] The systems and methods described herein enable the formation of small, intact cell patches (e.g., microtissues) and their application to a patient's body. Allowing these microtissues to release heat from the substrate on which the cell patches are cultured (e.g., an extracellular matrix protein scaffold (ECM)) allows the cells to maintain their structure and phenotype, and after delivery, to repair damaged tissue. The extracellular matrix surrounds the cultured cell patch (e.g., a monolayer of cells) and is configured to protect the cell patch from physical stress during the delivery process to the body. The ECM also provides binding sites that facilitate the binding of the cell patch to the desired target tissue (e.g., for tissue repair).

[0006] ECMs can include inclusion materials that replicate a desirable microenvironment for a cell patch in vivo. For example, an ECM can be configured to have a density, structure, and / or composition similar to that of a natural ECM, and these cells are surrounded in vivo. ECMs provide a unique microenvironment that more closely matches what is present in vivo, thus improving the ability to modulate cell behavior.

[0007] Cell patches can include cell monolayers. These monolayers are formed to contain cell expressions for tight cell junctions within the cell patch. Relatively large substrates and long culture times (e.g., about 24 hours) allow for the formation of tight junctions. These junctions generally occur in vivo, allowing the cell patch to be more readily accepted into tissues upon administration.

[0008] Shrink-packaged monolayers offer one or more of the following advantages: The biological characteristics of the cells are maintained for in vivo administration, in contrast to enzyme-based methods that release cells from the scaffold. The cells maintain their phenotype (e.g., tight junctions) within the monolayer. The cells form epithelial patches and / or construct a cytoskeleton that can be maintained for cell administration. When introduced in vivo in various contexts (e.g., in the heart, cornea, or for lung repair), the cells are readily taken up into tissues, as described below. Specifically, intercellular junctions are maintained.

[0009] As will be described later, cell patches can be used to treat diseases. For example, corneal repair can be performed using a cell monolayer. Cardiac repair can be performed. In some embodiments, lung repair can be performed.

[0010] As will be discussed later, monolayers offer other advantages over single-cell techniques. For example, endothelium and / or epithelium are barriers for cell administration. Since the administered cell monolayer is a foam cell patch, the endothelium allows the patch to be taken up. In some embodiments, the cell patch binds together as a dome of cells, flattens, and integrates with the endothelium over time. In addition, monolayer cells migrate from patch to other endothelium, which does not occur when individual cells are applied. Individual cells are less likely to bind. Therefore, monolayers have a higher viability compared to single cells. Intercellular binding can influence cell signaling and thus promote cell adhesion, maintaining the cell phenotype and increasing viability.

[0011] In some embodiments, cell addition materials are added to the extracellular matrix to facilitate patterning. For example, antibodies can be added to deliver to specific locations (e.g., targeted delivery). In some embodiments, growth factors (e.g., pbGF for muscle cells) can be added to the shrink-packaged matrix. This can enhance, for example, intravascular growth required by muscle cells. In some embodiments, this method includes labeling for validation. For example, cell tracking factors (e.g., cytoplasmic dyes) can be used.

[0012] In some embodiments, scaffolds of different shapes are possible. In addition to squares, tubes can be produced to create tubular structures. This can promote the growth of vascular fragments and / or chains of endothelial cells. In some embodiments, it is possible to mix different types of shrink-packaged cells.

[0013] Shrink-packaged cells can be used to treat a variety of diseases. For example, in some embodiments described herein, this includes injecting cells into patients suffering from corneal blindness due to low cell density. In some embodiments described herein, this includes micro-monolayer injections that can increase cell density without the need to remove existing endothelium.

[0014] In some embodiments, as described herein, the integration of a micro-monolayer of corneal endothelial cells that can be injected through a small bore to help mitigate density-dependent corneal blindness.

[0015] In some embodiments, those described herein include sequences of cell aggregates that can be produced by extracellular matrix shrinkage-packaged cell (SHELL) technology, in which, upon heat release, the cell aggregates can maintain their intercellular junctions and shrink to a size small enough to be injected into tissue through a needle.

[0016] In some embodiments, as described herein, the process involves an in vivo injection process that requires the injected eye to rest after injection to allow the cell aggregate to be taken up by the corneal endothelium. In some embodiments, as described herein, the process involves the synthesis of an extracellular matrix SHELL scaffold that can help maintain cells in a monolayer, maintain the cytoskeleton of cells in a monolayer, and improve integration along with their intercellular junctions.

[0017] In one embodiment, a method for encapsulating cells in microtissue comprises coating a tissue scaffold stamp with an extracellular matrix compound. The method comprises placing the tissue scaffold stamp on a thermoresponsive substrate. The method comprises seeding a cell culture onto the tissue scaffold stamp. The method comprises incubating the cell culture on the tissue scaffold stamp at a specified temperature, where the cell culture forms a cell patch that binds to the extracellular matrix compound. The method comprises forming a monolayer on the tissue scaffold stamp by the cell patch, where the monolayer boundary maintains expression for intercellular junction. The method comprises removing the thermoresponsive substrate. The method comprises removing the tissue scaffold stamp from the cell patch to form a microtissue structure around the cell patch. The method comprises folding the microtissue structure by suspending the microtissue in a solvent. The method comprises recovering the folded microtissue structure from the solvent. The method comprises administering the folded microtissue structure to a living organism.

[0018] In some embodiments, the method includes forming a tissue scaffold into a tubular structure. In some embodiments, the method includes forming tubular cell patches based on the tubular structure of the tissue scaffold.

[0019] In some embodiments, the cell patch contains a fragment of a blood vessel. In some embodiments, the method includes adding an antibody to the cell patch. In some embodiments, administering the microtissue structure to a living organism includes injecting the microtissue structure. In some embodiments, the cell patch contains corneal endothelial cells. The method includes introducing the cell patch into the cornea and using gravity to ensure contact between the cell patch and the cornea.

[0020] In some embodiments, the size of the microstructure is proportional to the size of the tissue scaffold stamp, where the size of the microstructure is part of the diameter of the injection device. In some embodiments, the tissue scaffold stamp contains an organosilicon compound. In some embodiments, the organosilicon compound contains polydimethylsiloxane. In some embodiments, the extracellular matrix compound contains a protein containing one or more of collagen IV, laminin, fibroblast growth factor protein, and vascular endothelial growth factor protein.

[0021] In some embodiments, the placement of the tissue scaffold stamp includes printing the tissue scaffold stamp onto a thermoresponsive substrate. In some embodiments, the thermoresponsive substrate contains a PIPAAm polymer. In some embodiments, the tissue scaffold stamp forms a regular shape. In some embodiments, the tissue scaffold stamp has a surface dimension of less than or approximately equivalent to 250 μm 2 and contains. In some embodiments, the cell patch contains 10 to 100 cells.

[0022] In a general aspect, the system includes a cell patch containing a cell monolayer that maintains the expression for intercellular junctions in the cell patch and the cytoskeleton of the cells. The system includes a microstructure folded around the cell patch and containing an extracellular matrix configured to provide a physical barrier between the cell patch and the external environment.

[0023] In some embodiments, the extracellular matrix contains a protein containing one or more of collagen IV, laminin, fibroblast growth factor protein, and vascular endothelial growth factor protein. In some embodiments, the microstructure forms a tubular structure. In some embodiments, the monolayer contains 10 to 100 cells. In some embodiments, the cell patch contains muscle tissue. In some embodiments, growth factors are added to the extracellular matrix to promote the internal growth of blood vessels in the muscle tissue.

[0024] Details of one or more embodiments of these systems and methods are described in the accompanying drawings and the description presented. Other features, purposes, and advantages of these systems and methods will become apparent from the specification and drawings, as well as the claims. [Brief explanation of the drawing]

[0025] [Figure 1A] This document describes a method for encapsulating the extracellular matrix. [Figure 1B] This document describes a method for encapsulating the extracellular matrix. [Figure 2] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 3] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 4] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 5A] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 5B] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 5C] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 6A] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 6B] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 7] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 8] This image shows extracellular matrix encapsulation using a tubular scaffold. [Figure 9] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 10] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 11A]Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 11B] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 12] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 13] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 14] Images and graphs of extracellular matrix encapsulation for monolayer corneal endothelial cell cultures are shown. [Figure 15] Figure 15 shows the method of extracellular matrix encapsulation and administration. [Modes for carrying out the invention]

[0026] The extracellular matrix (ECM) described herein comprises an array of geometric shapes that fold upon release. For example, the ECM can form one or more patterned shapes having dimensions of micrometers in length and width and nanometers in thickness. In some examples, the geometric shapes in the array include extracellular matrix proteins that can be used to culture cells, enabling the formation of 2D microtissues, particularly cell monolayers. These microtissues can then be released from the substrate once they have formed. In some embodiments, the microtissues are released thermally. Upon release, these microtissues spontaneously fold so that the ECM forms an outer layer around the cultured cells, and the cultured cells retain their microtissue structure and phenotype. The folded microtissues can then be administered, for example, by injection through a needle, to modify or replace the tissue. Microtissues have in vivo and in vitro applications. For example, microtissues can help form corneal endothelial cell microtissues that can be injected through a small-bore needle.

[0027] The methods and systems described herein involve the encapsulation of micromonolayer cell patches (e.g., μmonolayers) in a thin layer of ECM proteins, enabling cells to maintain high viability, intercellular junctions, and cytoskeletal structure after injection, while simultaneously imparting cell-ECM interactions that can promote cell adhesion. In vitro results confirmed that CE cells formed monolayers on a modified ECM substrate, constructed tight junctions within 24 hours, and formed an organized F-actin cytoskeleton structure, which was preserved throughout the heat release and injection process. Compared to enzymatically released single cells, the injected shrink-packaged μmonolayers significantly increased the CE monolayer density, as described later.

[0028] In addition, the shrink-packaged μ monolayers were bound to both in vitro and ex vivo corneas within 3 hours, demonstrating that the described encapsulation method can be applied using protocols recently used in clinical trials of single-cell CE injection. For example, an in vivo rabbit study using the eyes of healthy rabbits showed that a large number of shrink-packaged μ monolayers were incorporated into healthy CE. Generally, cells in CE of young, healthy rabbits are contact-blocked and present at extremely high densities. Therefore, if the shrink-packaged μ monolayers could integrate within such tissue, a very high rate of incorporation into damaged or affected CE would occur at very low cell densities.

[0029] Generally, the extracellular matrix (ECM) comprises a fibrous network of proteins, glycosaminoglycans, and other biomolecules. The ECM forms a pericellular skeleton that provides, for example, structural support, growth factor sequestration, adhesion, and mechanical signaling networks, as well as numerous other functions. The ECM can function as a suitable environment or niche for the function of cultured cells in a microtissue. For example, an adult stem cell niche contains specific ECM protein structures, compositions, supporting cell populations, and a set of soluble and insoluble signaling molecules that help maintain the pluripotency of stem cells. The ECM is not a naturally occurring ECM, but rather an artificially produced protein substrate. The selection of ECM proteins is based on the cell culture being produced, as will be further detailed below.

[0030] In some cases, 2D cultured cells are typically grown on hard tissue culture-treated polystyrene (TCPS) that is pre-coated with ECM proteins or coated with ECM proteins contained in serum supplemented into the medium. While such ECM proteins enable cell adhesion to TCPS and subsequent proliferation, many primary cell types can only pass through for a limited time before their phenotype changes due to aging, such as epithelial-mesenchymal transition (EMT). 3D cultures using synthetic and / or native hydrogels can address some of these limitations by altering the chemodynamic environment to better replicate in vivo conditions and have been effective for culturing a wide range of cell types. However, these hydrogels are typically structurally isotropic and do not replicate ECM-enriched structures such as basement membranes, and have compositions that are typically different from those of complexes in the in vivo environment (e.g., collagen, fibrin, Matrigel, PEG). Furthermore, in both 2D and 3D cultures, the passage of these cells often requires the use of enzymes and calcium chelators that cleave cell-matrix and cell-cell adhesions to produce single-cell suspensions. When reseeded, cells must expend energy to reconstruct the cell matrix and intercellular adhesions within the new environment in which they are placed. ECMs are configured to mimic the cellular microenvironment found in vivo by (i) encapsulating cells in a predetermined ECM that better mimics the natural ECM structure, and (ii) minimizing cell-matrix and intercellular adhesions through division.

[0031] ECM nanoscaffolds are formed that can be used to at least partially encapsulate cells in order to modulate the chemodynamic microenvironment. Using surface initiation aggregate (SIA) adaptations, distinct nanoscaffolds of organized ECM proteins are formed into self-supporting structures. Cells are encapsulated (e.g., shrink-packaged) within the organized protein matrix layer by adhering them to these ECM nanoscaffolds before release. In some embodiments, the ECM nanoscaffolds are modified to a cell-scale size with a lateral dimension of approximately 75 μm and a thickness of approximately 50 nm. In some embodiments, the SIA method can be used to encapsulate various cell types within a given ECM containing one or more of the major protein compositions of the native pericellular matrix: fibronectin (FN), laminin (LAM), fibrinogen (FIB), and collagen type IV (Col IV). The long-term goal is for these ECM nanoscaffolds and encapsulation methods to enhance the delivery of therapeutic cells by helping to integrate cell survival and function in otherwise impaired matrix environments, such as those found in infarcted cardiomyocytes.

[0032] ECM nanoscaffolds can be used with any adherent cell type, and even with non-adherent cells, provided that antibodies for cell surface markers are mixed in the ECM protein solution before incubation on the PDMS stamp. For example, cell types include hepatocytes, which are adherent cell types. For example, cell types include killer T cells, which are non-adherent cell types, and can be combined with cell surface marker antibodies in the ECM. These cell types can be used with collagen I, collagen IV, fibronectin, laminin, vitronectin, and any ECM proteins that can be microcontact printed.

[0033] Figure 1A shows an exemplary method 100 for shrink-packaging and injecting a corneal endothelial encapsulated cell patch (e.g., a cell monolayer). Using a surface-initiated assembly technique, a 200 micrometer (μm) × 200 μm × 5 nanometer (nm) ECM scaffold is modified on a thermoresponsive polymer PIPAAm. The sample and cells are then heated to 40°C, after which the cells are seeded on the square and cultured for approximately 24 hours. After approximately 24 hours, the sample is rinsed in a warm medium and cooled to room temperature to induce dissolution of the PIPAAm and shrink-packaging / release of a micron-sized monolayer of cell patch (e.g., corneal endothelial cells).

[0034] Method 100 can be implemented for each of the following embodiments.

[0035] ECM scaffolding generation ECM scaffolds were generated via the aforementioned surface-initiated assemblies with minor modifications (e.g., shrink wrapping paper). Briefly, a 1 cm × 1 cm PDMS stamp designed to have a 200 μm × 200 μm square feature was fabricated using standard soft lithography techniques. The stamps were sonicated in 50% ethanol for 60 minutes, dried under a nitrogen stream, and incubated for 60 minutes with a 50:50 mixture of 50 μg / mL collagen IV (COL4) and 50 μg / mL laminin (LAM), as shown in step 110. Proteins were visualized using either 50% AlexaFluor488-labeled COL4 or 50% AlexaFluor633-labeled LAM. After incubation, the stamp was rinsed with sterile water, dried under a nitrogen stream, and subjected to isometric contact for 30 minutes with a 25 mm glass coverslip coated with poly(N-isopropylacrylamide) (PIPAAm) (2% high molecular weight, Sceintific Polymers) to ensure the movement of the square as shown in step 120. An ECM square microcontact printed on a PDMS coverslip was used as a control. Upon removal of the stamp, the quality of the moved ECM square was determined using laser scanning confocal microscopy (Nikon AZI00).

[0036] Example of embodiment: Bovine corneal endothelial cell culture Bovine corneal endothelial cells (BCECs) were isolated and cultured as described above (ref EBM and expanded paper). Briefly, corneas were excised from eyeballs (Pel Freez) and incubated endothelial-side up in a ceramic 12-well spot plate with 400 μL of TrypLE Express for 20 minutes. The cells were then carefully scraped from the cornea using a rubber spatula, centrifuged at 1500 RPM for 5 minutes, and resuspended in 5 mL of medium (specified as PO, low glucose DMEM with 10% FBS, 1% Pen / Strep / AmphB, and 0.5% gentamicin, cultured in T-25 flasks coated with PDMS at 50 kPa). Fifty eyeballs were obtained at once and used to seed five T-25 flasks. The cells were cultured until confluent and divided 1:3 until confluent P2 cells were used at once.

[0037] ECM scaffolding with shrink-cut BCECμ single layer A patterned coverslip (25 mm) was secured to the bottom of a 35 mm Petri dish with vacuum grease and placed on a dry block set to 52°C. This allowed the coverslip to reach and maintain a temperature of 40°C (within 30 minutes). Bovine CEC was released from a culture flask with TrypLE Express, centrifuged, and resuspended in a 15 mL centrifuge tube at a density of 150,000 cells / mL. The tube was placed on a dry block set to 45°C for approximately 5 minutes, or until the cell solution reached just 40°C. After adding 2 mL of cell suspension to each 35 mm dish, it was immediately placed in an incubator (37°C, 5% CO2). The cells were cultured on a square for 24 hours to form a small monolayer on a 200 μm square. After 24 hours, the sample was removed from the incubator, rinsed twice with medium at 37°C, 2 mL of fresh warm medium was added, and the sample was allowed to cool to room temperature. Once the temperature dropped below 32°C, PIPAAm dissolved, releasing the scaffold + μ monolayer. The release process was recorded using a Photometrics CoolSnap camera. After release, the scaffold + μ monolayer was recovered by centrifugation at 150 rpm for 5 minutes before use in further experiments. CEC seeded on PDMS coverslips was used as a control.

[0038] Immunostaining of shrink-wrapped BCEC: Shrink-wrapped μ monolayers, resuspended in PBS containing Ca2 and Mg2 (PBS++), were injected onto glass coverslips through a small-diameter needle and fixed for approximately 15 minutes, followed by fixation in 4% paraformaldehyde (PBS++) for 15 minutes. The samples were carefully washed twice with PBS++ and incubated with DAPI 1:100 dilution, mouse anti-ZO-1 antibody (Life Technologies) 1:100 dilution, and AlexaFluor488 3:200 dilution. The samples were rinsed twice with PBS++ for 5 minutes and incubated with AlexaFluor555 goat anti-mouse secondary antibody 1:100 dilution for approximately 2 hours. The samples were rinsed twice with PBS++ for 5 minutes, placed on glass slides with Pro-Long Gold Antifade (Life Technologies), and then imaged with a Zeiss LSM700 confocal microscope.

[0039] Survival rate after shrink-wrapped BCEC injection After centrifugation, shrink-packaged μ monolayers or TrypLE Express-released single cells were resuspended in 200 μL of culture medium, drawn into a 280 needle, injected into a Petri dish, and incubated at 37°C for 30 minutes with 2 μM calcein AM and 4 μM EthD-1 (Live / Dead Viability / Cytoxicity Kit, Life Technologies) in PBS++. After 30 minutes, samples were imaged with a Zeiss LSM700 confocal microscope, using 5 images per sample and 3 samples per type. The number of viable and dead cells was manually counted. The number of viable cells was divided by the number of dead cells to determine the % viability of both ECM scaffold-packaged cells and enzymatically released cells.

[0040] Seeding of shrink-wrapped BCEC and single BCEC on interstitial mimics Self-compressing collagen I membranes were prepared as described above. Briefly, a 6 mg / mL collagen I gel solution was prepared according to the manufacturer's instructions and pipetted into a 9 mm diameter silicone ring at the top of a glass coverslip. The gel was placed in a wet incubator (37°C, 5% CO2) for 3 hours to compress under its own weight. The gel was then completely dried in a biofood and rehydrated in PBS to form an ultrathin collagen I stromal mimetic. Shrink-w packaged BCEC μ monolayers were seeded onto the membrane in a 1:1 ratio of stamped coverslips to collagen I membranes. As a control, BCEC cultured in a flask and BCEC enzymatically released into single-cell suspensions using TrypLE Express were seeded onto the collagen I membranes. In the best-case scenario, where all squares had full monolayers, the number of seeded control cells was equal to the number of cells that would have been seeded from one stamped ECM scaffold sample. The average number of cells occupying a 200 μm square was 30, therefore, 30 cells × 1600 squares per stamp, meaning approximately 48,000 cells per sample. Thus, 50,000 cells per sample were seeded as a control. At 6 and 24 hours, the samples were removed from the culture medium, fixed, and stained for nuclear ZO-1 (tight-junction protein) and F-actin.

[0041] In short, the samples were rinsed twice with PBS++ and fixed with 0.05% Triton-X100 in 4% paraformaldehyde (in PBS++) for 15 minutes. The samples were rinsed twice with PBS++ for 5 minutes each and incubated with 5 drops of NucBlue (Life Technologies) for 10 minutes. The samples were rinsed once with PBS++ and incubated for 2 hours with a 1:100 dilution of mouse anti-ZO-1 antibody (Life Technologies) and a 3:200 dilution of AlexaFluor488 or 633 phalloidin. The samples were rinsed three times with PBS++ for 5 minutes each and incubated for 2 hours with a 1:100 dilution of AlexaFluor555 goat anti-mouse secondary antibody. The samples were rinsed three times with PBS++ for 5 minutes each, placed on a glass slide using Pro-Long Gold Anti-fade, and imaged with a Zeiss LSM700 confocal microscope.

[0042] In vitro integration of shrink-wrapped CEC versus single-bovine CEC. To mimic low-density aging CE, 25,000 PS bovine cells were seeded onto the above-described collagen I stromal mimetic until confluent to form “aged” monolayers. Shrink-packaged μ monolayers and monoBCEC were prepared as described above, labeled with CellTracker Green (Life Technologies) for 30 minutes, centrifuged, diluted to the equivalent of 50,000 cells / sample, and injected onto the “aged” monolayers. “Aged” monolayers without cell injection at the apex served as controls. Samples were stained and fixed on days 3, 7, and 14 as described above. Ten random spots were imaged on each sample using a Zeiss LSM700 confocal, and cell density was manually counted (e.g., cell nuclei). The number of nuclei was divided by the image area to obtain cells / mm2 per image. The cell density for each sample was determined by averaging the cell density for each image, and the average cell density for each sample type was determined by averaging the cell densities of 3 or 4 samples. To investigate the temporal external growth of shrink-wrapped μ monolayers, central confocal images and individual shrink-wrapped μ monolayers (day 3, n=33; day 7, n=37; day 14, n=40) were collected, and the CellTracker channels were converted to binary black and white images. The binary images for each sample type were then converted to Z-stacks, and the average pixel density of the CellTracker was determined by analyzing the Z-stack plugin (relative values ​​without log10) using Heat Map.

[0043] Live imaging of in vitro integration of shrink-wrapped bovine CEC. For live imaging, the “aged” monolayer on a collagen I stromal mimetic was first incubated for 30 minutes using CellTracker Orange to distinguish between the existing monolayer and the injected cells labeled with CellTracker Green as described above. HEPE-buffered Opti-MEM I Reduced Serum Media (Life Technologies) containing 10% FBS and 1% Pen / Strep was added to the monolayer, and the shrink-wrapped μ-monlayer prepared as described above was injected into the top of the sample using a 30G needle. The sample was placed on a Zeiss LSM700 confocal with a temperature chamber set to 37°C for 30 minutes to allow cell fixation. A time-lapse series of one z-stacks was obtained hourly for 48 hours using the Definite Focus system.

[0044] Rabbit CEC isolation, culture, and shrink packaging Rabbit eyeballs were received on ice from Pel Freez Biologicals. The corneas were excised from the eyeballs, and the CE and Descemet's membranes were manually peeled off using forceps. The cells were incubated in Dispase (1 U / mL, Stem Cell Technologies) for 1.5 hours at 37°C to detach the rabbit CE cells (RCEC) from the Descemet's membranes. The RCEC cells were then carefully pipetted up and down, diluted in medium (DMEM / Fl2, 10% FBS, 0.5% Pen / Strep), centrifuged at 1500 RPM for 5 minutes, resuspended in 10 mL of medium, specified by PO, and cultured in COL4-coated T-25 flasks, each containing 15–25 eyes depending on the cell yield. The RCEC cells were cultured until confluent, divided 1:2, and used in all experiments once confluent in P1 or P2. The RCECμ monolayer was shrink-packaged with the following modifications as described above: an ECM scaffold (using the same 1cm x 1cm PDMS stamp) was stamped onto an 18mm glass coverslip to avoid having an excessive seeding area and to reduce the number of cells that needed to be seeded per sample so as to still achieve a complete coverage of approximately 50,000 cells / sample once confluence was reached. The coverslip was fixed to the bottom of a Nunc IVF center well dish (20mm inner diameter well) with vacuum grease, the cells were resuspended at 150,000 cells / mL, and 1mL of RCEC was seeded per sample.

[0045] Exvivo integration of shrink-wrapped rabbit CEC Three rabbit eyeballs were placed in a 12-well plate with the cornea facing upward. Shrink-packaged RCECμ monolayers were prepared as described above. Two μ monolayer samples were prepared per ex vivo eye and resuspended in 100 μL of DMEM / Fl2. Using a 30-G insulin syringe, the total 100 μL was drawn up, and the needle was inserted into the center of the cornea until visible in the anterior chamber, and 50 μL of the suspension was injected. This injected cells equivalent to 50,000 cells into the anterior chamber. The needle was held in place for several seconds to ensure that the medium and cells did not return outside the injection site. The injection was observed under a stereomicroscope, and the pink color of the medium filling the anterior chamber was visible to the naked eye, indicating a successful injection. The eyes were inverted with the cornea facing downward and incubated at 37°C for 3 hours under 5% CO2 in a humidified incubator. After 3 hours, the eyeballs were placed in 2% paraformaldehyde (PBS++) at 4°C for 24 hours. After 24 hours, the eyes were rinsed with PBS, the corneas were extracted, and rinsed three times for 5 minutes each. The corneas were then incubated at room temperature for 2 hours on 1 mL of PBS++ containing 2 drops of NucBlue (Life Technologies), a 2:100 dilution of mouse anti-ZO-1 antibody (Life Technologies), and a 3:200 dilution of AlexaFluor488 phalloidin (Life Technologies), with the CE side facing down. The corneas were then rinsed three times in PBS for 5 minutes each, and subsequently incubated for 2 hours on 1 mL of PBS++ containing a 2:100 dilution of AlexaFluor555 goat anti-mouse secondary antibody, and stored in PBS before imaging with a Zeiss LSM700 confocal microscope.

[0046] Shrink-packaged CEC for in vivo injection and integration Shrink-packaged RCECμ monolayers were prepared as described above with one minor modification: cells were incubated in 1 mL of medium containing 5 μL of Vybrant DiO for 30 minutes, and then labeled with Vybrant DiO the day before seeding onto ECM nanoscaffolds by rinsing three times in fresh medium for 10 minutes each. An excess number of μ monolayer samples were prepared to ensure a sufficient dose for injection (16). The shrink-packaged μ monolayers were released and resuspended in a total of 300 μL in one 1.5 mL microcentrifuge tube. Three healthy, intact New Zealand White female rabbits weighing approximately 2.5 kg were used in this study. The rabbits were anesthetized with ketamine (40 mg / kg) and xylazine (4 mg / kg), and then sedated for 3 hours using isofluorene. Rabbit number 1 was injected into the right eye (OD) with 50 μL (approximately 100,000 cells). Rabbit number 2 was initially injected with 100 μL into its right eye, but most of the volume returned outside the cornea. Therefore, an additional 50 μL was injected into a second location, ensuring that approximately 300,000 cells were injected if all cells successfully remained in the anterior chamber. Rabbit number 3, having a smaller-than-average right eye, was injected with 100 μL or approximately 200,000 cells into its left eye. Immediately after injection, each rabbit was placed with the injected eye facing downwards for 3 hours to ensure cell colonization. On day 7, the rabbits were anesthetized by intramuscular injection of ketamine (40 mg / kg) and xylazine (4 mg / kg), and then euthanized by intravenous injection of Euthasol solution (1 mL / 1.8 lbs) containing 390 mg / mL pentobarbital sodium and 50 mg / mL phenytoin sodium. The eye was immediately enucleated, and 100 μL of 2% paraformaldehyde (PBS++) was injected intravitreously.

[0047] The eyeballs were then immersed in 2% paraformaldehyde (PBS++) and fixed at 4°C for 24 hours. After 24 hours, the eyes were rinsed with PBS, the corneas were extracted, and rinsed three times over 5 minutes. The corneas were then incubated at room temperature for 2 hours on 1 mL of PBS++ containing 2 drops of NucBlue (Life Technologies) and a 2:100 dilution of mouse anti-ZO-1 antibody (Life Technologies), with the cerebrospinal fluid (CE) facing downwards. The corneas were then rinsed three times in PBS over 5 minutes, and subsequently incubated for 2 hours on 1 mL of PBS++ containing a 2:100 dilution of AlexaFluor555 goat anti-mouse secondary antibody, and stored in PBS before imaging with a Zeiss LSM700 confocal microscope.

[0048] Example Embodiment: Capillary ECM Structure of Cardiac Tissue In one embodiment, tubular endothelial portions are generated by culturing endothelial cells on micropatterned fibronectin rectangles on PIPAAm (a thermosensitive polymer). After lysis of PIPAAm, the tubular portions are released and can be incorporated into cultured cardiac tissue to create a primitive vascular network. The fibronectin rectangles are stamped onto PIPAAm. Endothelial cells are seeded on three different stamping patterns, forming confluent monolayers on the rectangles at different seeding densities. Thermal control is used to release the endothelial tubules from the PIPAAm surface.

[0049] The tubular endothelium is constructed for the treatment of cardiovascular disease (CVD). CVD is a major global concern and a leading cause of death worldwide. CVD includes myocardial infarction, heart failure, and congenital heart defects, which can lead to a decline in the heart's ability to supply blood to tissues throughout the body. Recent advances in tissue engineering have allowed for the creation of three-dimensional cardiac tissue from fibroblasts in combination with cardiomyocytes derived from embryonic stem cells or induced pluripotent stem cells. These cultured cardiac tissues could potentially be used to "patch" damaged areas of the heart. However, the heart is an extremely complex organ with multiple cell types in addition to fibroblasts and cardiomyocytes. The cell patches described herein are constructed to provide a supply of vascular tissue.

[0050] Figure 1B shows method 105 for forming endothelial tubular portions. The endothelial tubular portions are generated in three dimensions and can be incorporated into cultured cardiac constructs. This is thought to increase the size of grafts produced by growable tissue engineering, which may have a greater ability to repair damaged hearts. The dimensions of the micropatterned rectangles and the concentration of HUVEC for producing the most effective 3D endothelial tubular portions were determined as follows: Fibronectin rectangles were micropatterned on poly(N-isopropylacrylamide) (PIPAAm), a thermosensitive polymer. Endothelial cells were then cultured on these fibronectin rectangles to form tight junctions, which are important intercellular connections found in tissue vascular systems. The tubular portions, consisting of fibronectin and endothelial cells, were then released after PIPAAm lysis.

[0051] As shown in Figure 1B, in step 115, the PEMS stamp is coated in labeled fibronectin and then dried. In step 125, the PDMS stamp is placed stamp-side down on a PIPAAm-coated glass coverslip to bond the ECM-coated stamp. In step 135, after removing the stamp, HUVEC is seeded onto the coverslip and bonded at 37°C for 24 hours. In step 145, a medium at room temperature is added to the coverslip to dissolve the heat-sensitive PIPAAm. In step 155, the ECM rectangle is released and packaged around the cells. Each of these steps will be described in detail below.

[0052] Spin coating of PDMS and PIPAAm onto glass cover slips Glass coverslips were sonicated in a 50% v / v ethanol solution to remove all particulate matter. The coverslips were then dried using a nitrogen spray gun and placed in a large Petri dish. The PDMS used for spin-coating these coverslips was prepared by using Sylgard 184 and a curing agent in a 1:10 ratio. This solution was then mixed until homogeneous, placed in a vacuum chamber, and defoamed for 45 minutes. A 200 μL droplet of PDMS was then placed in the center of each coverslip, and the coverslip was added to the spin coater. The coverslips were spin-coated at 2000 rpm for 2 minutes. The PDMS-coated coverslips were cured at 65°C for 4 hours. Once completed, the coverslips were stored until further use.

[0053] HUVEC culture Remove the HUVEC from the liquid nitrogen chamber, thaw it in a water bath, combine it with 10 mL of culture medium, allow it to settle on a pellet, then aspirate it and combine it with more culture medium, for a total of 10 mL 6 The concentration was increased to cells / mL. Then the cells were added at a rate of 5000 cells / cm³. 2 At this concentration, 175cm 2 The cells were distributed into flasks. The culture medium was changed every other day until the cells reached approximately 80% confluence (at which point the experiment was conducted).

[0054] Stamping onto a fibronectin-coated coverslip First, fibronectin was prepared by creating a 50 μg / mL unlabeled fibronectin solution. This was done by combining 3.8 mL of deionized water with 0.2 mL of 1 mg / mL unlabeled fibronectin. From this, a 10% labeled fibronectin solution was prepared by combining 900 μL of the 50 μg / mL unlabeled fibronectin solution, 92.5 μL of deionized water, and 7.5 μL of 667 μg / mL labeled 633-fibronectin.

[0055] The PDMS stamps were placed in a beaker filled with 50% ethanol solution and 50% distilled / deionized water. This beaker was placed in an ultrasonic generator for at least 30 minutes. The stamps were then individually picked up using sterile tweezers and dried using a nitrogen gun. The stamps were then placed in a sterile petri dish and allowed to await fibronectin coating. Then, as shown in step 115, 300 μL of fibronectin solution was pipetteed onto each PDMS stamp with the stamp facing upwards. Using a pipette, the fibronectin was spread over the entire area of ​​all four corners of the PDMS stamp, and the stamps were allowed to coat for at least 1 hour.

[0056] Near the end of this hour, the PDMS-coated coverslips were placed in the UVO cleaner for 15 minutes, with the lids positioned under the dish inside the cleaner. Immediately after 15 minutes, the petri dishes were placed in the biofood to maintain sterility. Using tweezers, each coverslip was placed into its own well on a 6-well culture plate.

[0057] The PDMS stamps were washed individually by stirring in a dish of deionized water, and then washed again in another dish of deionized water. The stamps were then dried using a nitrogen gun, and then, as shown in step 125, the stamps were turned upside down onto the PDMS-coated coverslip using a second pair of forceps to ensure good contact. The stamps were gently tapped to ensure good contact with the coverslip. The stamps were then lifted from the coverslip without twisting so as not to damage the transferred pattern. The coverslips were then immersed in 1X PBS for preservation purposes.

[0058] The protocol for stamping coverslips coated with PIPAAm differs from the protocol described above only in certain steps. This includes removing the lid of the petri dish during UVO treatment and not using PBS for preservation. Generally, the stamp should remain on the coverslip for at least 45 minutes and up to 24 hours.

[0059] Seed sowing on PDMS stamp cover slips As shown in step 135, aspirate the PBS from the stamped coverslip, then fill with 150,000 cells / cm³. 2 , 300,000 cells / cm 2 , and 450,000 cells / cm² 2 Seeds were sown at the HUVEC concentration.

[0060] Sowing on PIPAAm stamp coverslip In one embodiment, in step 135, the heat block was sprayed with 70% ethanol and placed in a biofood. The left side was set to 52°C and the right side to 45°C. Stamped PIPAAm coverslips were secured to the bottom of their own petri dishes using vacuum grease. The petri dishes were then placed on the left side of the heat block. Each coverslip required 2 mL of fluid. The intended experimental cell concentrate was added to a 15 mL tube and placed on the right side of the heat block for approximately 5 minutes or until a temperature of 38–40°C was achieved. The coverslips reached a temperature of 38–40°C. Then 2 mL of cell solution was added to each coverslip and immediately placed in an incubator to prevent any temperature drop that might prematurely dissolve the PIPAAm.

[0061] The substrate releases heat to form endothelial tubules. In step 145, PIPAAm was heated through the process of adding room temperature culture medium to a dish under a microscope. The medium was aspirated to fix the cells, washed with 1X PBS containing calcium and magnesium, aspirated again to ensure no cells were aspirated, then 4% formaldehyde was added in a hood, waited 30 minutes, at which point the solution was carefully aspirated, the cells were immersed in 1X PBS and then stored.

[0062] The coverslip was the primary ZO-1 antibody (5:200 ratio), and then washed three times with 1x PBS (with 30-minute intervals in between). The coverslip was then stained with DAPI (1:100 ratio), 488 phalloidin (3:200 ratio), and secondary goat anti-mouse 555 (5:200 ratio). The ZO-1 antibody stains for adherens junctions between cells. These adherens junctions are an indicator of a high degree of cell confluence and are important in the formation of endothelial tube segments.

[0063] Referring to FIG. 2, images 200a - c show fibronectin stamped on a PDMS-coated coverslip having the following dimensions. In image 200a, the dimensions include a 200×10 μm rectangle. In image 200b, the dimensions include a 200×20 μm rectangle. In image 200c, the dimensions include a 200×30 μm rectangle. The scale bar for each of these images is 50 μm. These examples show that it is possible to accurately pattern micropatterned fibronectin rectangles onto a PIPAAm substrate.

[0064] FIG. 3 shows images 300a - c of HUVECs seeded on a 200×10 μm stamp PDMS-coated coverslip at different cell concentrations. Image 300a shows a cell concentration of 2 150,000 cells / cm 2 Image 300b shows a cell concentration of 300,000 cells / cm 2The image shows the concentration of [the cell type]. Here, the scale bar in each image is 500 μm. Generally, human umbilical cord endothelial cells (HUVECs) at different seeding concentrations were used to evaluate the ability of endothelial cells to bind to micropatterned fibronectin rectangles and form a confluent monolayer on them within 24 hours. As the seeding concentration of HUVECs increased, the number of endothelial cells filling the gaps between rectangles increased. This was particularly pronounced in stamps where the rectangles were separated only by short distances of 10 μm (200 × 10 μm patterns). However, at higher cell concentrations, the rectangles became indistinguishable, indicating that 200 × 10 μm rectangles are too dense to seed at high concentrations of HUVECs.

[0065] Figure 4 shows images 400a-400c of HUVEC seeded on 200 × 20 μm stamped PDMS-coated coverslips at different cell concentrations. Image 400a shows a cell concentration of 150,000 cells / cm². 2 This shows the concentration. Image 400b shows 300,000 cells / cm³. 2 This shows the concentration. Image 400c shows 450,000 cells / cm³. 2 This shows the concentration. In images 400a-c, the scale bar is 500 μm. In contrast to images 300a-c, as cell concentration increases, there is more space (200 × 20 μm) between adjacent rectangles, but the overlap is minimal, and the rectangles maintain their distinguishable shape. The cells are very confluent, which is an aspect that helps activate the intima of the microvascular system in vivo.

[0066] Figures 5A–5C show confocal images 500a–c of HUVEC seeded on 200 × 10 μm stamped PDMS-coated coverslips at different cell concentrations. Image 500a in Figure 5A shows a cell density of 150,000 cells / cm². 2 This shows the cell concentration. Image 500b in Figure 5B shows a cell concentration of 300,000 cells / cm³. 2 This shows the cell concentration. Figure 5C shows 450,000 cells / cm³. 2The cell concentrations are shown. Coverslips were stained with DAPI (blue), phalloidin (green), and ZO1 antibody (red) for tight junction staining. Stamped fibronectin can be seen in darker chromaticity (e.g., magenta) in images 500a and 500c. In each image 500a-c, the scale bar is 50 μm.

[0067] As previously mentioned, tight junctions are important binding components present between endothelial cells in the tissue vascular system. To determine whether tight junctions are present in HUVECs seeded on micropatterned rectangles, an antibody targeting ZO-1 is used. This antibody is a marker of tight junctions. As shown in image 500a in Figure 5A, very low levels of ZO-1 were observed in samples with the lowest seeding density. This is due to low intercellular contact in low-density seeded samples. Tight junctions between cell boundaries are easily identifiable in the high-density monolayer in image 500b in Figure 5B. A "no primary antibody" control is shown in image 500c in Figure 5C to demonstrate the specificity of the antibody against ZO-1 tight junctions. The lack of stained tight junctions in image 500c indicates that the staining ZO-1 antibody was effective in image 500b.

[0068] Figures 6A-6B show confocal images 600a-b of HUVEC seeded on 200 × 20 μm stamped PDMS-coated coverslips at different cell concentrations. Image 600a in Figure 6A shows a cell density of 150,000 cells / cm². 2 This shows the cell concentration. Image 600b in Figure 6B shows 450,000 cells / cm³. 2 The cell concentrations are shown. Coverslips were stained with DAPI (blue), phalloidin (green), and ZO1 antibody (red) for staining tight junctions. In each image 600a-b, the scale bar is 50 μm. HUVEC at concentrations in both images 600a-b is highly confluent. This can be seen by the high abundance of staining tight junctions. Despite the increase in cell concentration, the cells are still able to maintain their distinct rectangular patterning.

[0069] Figure 7 shows 300,000 cells / cm². 2 Images 700a–f show the time periods before and after release of HUVEC seeded on a 200 × 20 μm stamp PDMS coated with the cell concentration. To test whether HUVEC seeded on a micropatterned rectangle could be released, the inventors patterned a 200 × 20 μm rectangle on a PIPAAm-coated coverslip and seeded HUVEC on it. Before release, the HUVEC seeded on the rectangle demonstrated high confluence and specific binding to the micropatterned rectangle. Each image 700a–f represents the same coverslip. In images 700a–f, the scale bar is 500 μm. As seen in image 700a, all corners of the rectangle have sharp contours, indicating how effective the seeding was in terms of complete coating of the fibronectin stamp. In images 700c-f, after PIPAAm emission, HUVEC was able to maintain its individual patterns, show signs of curling, and may have formed cylindrical portions in three dimensions.

[0070] Figure 8 shows 300,000 cells / cm³ released, stained, and imaged. 2 Images 800a–d show HUVEC seeded on 200 × 20 μm stamped PDMS-coated coverslips at the cell concentration. Each image 800a–d represents the same coverslip. The coverslips were stained with DAPI (dark) and phalloidin (light). Images 800a–c were taken at 10× magnification. Image 800d was taken at 20× magnification. The scale bar in each image is 50 μm. The confocal images of the released tubular portions show that the released rectangles maintain their individual parts and form tubular structures. These structures appear to curl slightly inward in each image 800a–d.

[0071] In one aspect, based on the aforementioned images, an example of an optimal cell concentration is approximately 300,000 cells / cm³. 2 Generally, 450,000 cells / cm³ 2The size was too large for all three micropatterning dimensions. The 200 × 20 μm micropatterning rectangle had the most confluent cells compared to the 200 × 10 μm size, which tended to extend beyond the micropatterning rectangle.

[0072] Example Embodiment: Packaging μ-layer of corneal endothelial cells The cerebrospinal layer (CE) is a monolayer of cells lining the posterior surface of the cornea, involved in maintaining appropriate corneal thickness and clarity by pumping excess fluid from the interstitium into the anterior chamber. CE cells are arrested during the GI phase of the cell cycle and therefore cannot replicate to repair damage from disease, trauma, or normal aging. As a result, when cells die, the remaining cells hypertrophy to maintain monolayer integration and pumping function, leading to a decrease in cell density. Cell density is approximately 500 cells / mm². 2 When the cerebrospinal fluid (CE) level drops below a certain point, the CE can no longer perform its function properly, leading to the accumulation of excess fluid in the corneal stroma and resulting in corneal blindness. The structure (cellular monolayer), function (barrier / fluid pumping), and location (adjacent to the anterior chamber) of the CE make it an ideal target for cell injection therapy compared to more complex tissues. Furthermore, animal models and clinical trials have shown that injection into the anterior chamber for CE repair suffers from a lack of cell retention, viability, and integration into the CE, requiring the injection of numerous cells and small molecules such as the ROCK inhibitor Y-27632 to achieve the desired results. However, even at best, its regenerative capacity is limited, particularly because the intercellular junctions and cytoskeletal structure of CE cells have been shown to be necessary for cell signaling, function, and maintenance of a mature, non-proliferative monolayer state through contact inhibition.

[0073] This embodiment describes the micron-scale shrink packaging (e.g., encapsulation) of CE cells (CECs) in a nanometer-thick ECM scaffold by the encapsulation method described with respect to Figure 1A. During shrink packaging, the micron-scale monolayer (μ monolayer) shrinks to a size small enough to be injected through a 300-gauge needle while maintaining a well-organized monolayer cell sheet with intercellular junctions and cytoskeletal structures. In vitro, shrink-packaged μ monolayers significantly increase the cell density of an existing monolayer compared to single-cell encapsulation.

[0074] This description explains experimental results demonstrating in vivo integration of shrink-packaged μ monolayers into the anterior chamber of rabbit eyes to increase cell density. The experiment showed that shrink-packaged CECμ monolayers in the ECM scaffold maintained the cytoskeletal structure, tight junctions, and viability of cells after injection.

[0075] To modify the CEC monolayer to micron-scale dimensions (μ monolayer), modifications were made to shrink-packaged single cells (referred to as SHELLs) in nanometer-thick ECM scaffolds. Here, the dimensions of the squares patterned on poly(N-isopropylacrylamide) (PIPAAm) by surface-initiated assembly technology were increased to approximately 200 μm × 200 μm. The increased scaffold size allows more cells to adhere to each scaffold. The culture time of cells on the squares, previously up to 24 hours, was extended to approximately 24 hours. Because PIPAAm is heat-responsive, the sample and cells were heated to 40°C during the seeding process to prevent PIPAAm from dissolving.

[0076] Refer to Figure 9 to see an example of a cultured cell monolayer. As a control, a scaffold of the same size was microcontact printed onto PDMS, as shown in Image 900a. CECs form monolayers on ECM squares microcontact printed on PDMS (used as a control) and on the thermoresponsive polymer PIPAAm. Once the PIPAAm dissolves, the CEC monolayer shrinks and shrinks within the ECM square. After 24 hours, bovine CEC (BCEC) μ monolayers cultured on ECM scaffolds made on PIPAAm showed a similar morphology to those cultured on ECM scaffolds made on PDMS when viewed under a phase-contrast microscope.

[0077] Image 900b shows that CEC release and shrink packaging occur immediately after the sample cools to room temperature, in less than 100 seconds. During the dissolution and heat release of PIPAAm, the μ monolayer remained in its original state and was successfully shrink-packed within the ECM square. After release, the shrink-packed μ monolayer was collected, centrifuged, injected through a 280 needle, fixed for 30 minutes, and then fixed and stained to examine the structure of BCEC within the μ monolayer. As seen in Image 900c, the shrink-packed BCEC exhibited continuous Z0-1 and cortical F-actin structures at the boundary, indicating that the cells maintained tight junctions and cytoskeletal structures throughout the release process. Confocal microscopy images show that CECs retain both cytoskeletal structures (F-actin) and tight junctions (Z0-1) after injection.

[0078] Image 900d includes a 3D projection of a shrink-packaged CEC monolayer 30 minutes after injection, illustrating the process of relaxation and return to its original shape. Image 900d shows that after release, the shrink-packaged μ monolayer begins to relax and returns to an engineered square-like structure approximately 30 minutes after injection. Cell viability in the μ monolayer after injection was examined using viability / dead cytotoxicity analysis and compared to single cells released enzymatically. Image 900e shows a representative viability / dead image of a control single CEC, showing that in the shrink-packaged CEC, both types of cells are viable, with very few dead cells present. Confocal microscopy images showed that only dead cells in the shrink-w-packaged μ monolayer sample were individual cells that had not been integrated into the μ monolayer.

[0079] Survival / death data (n=3) were statistically compared using Student's t-test, and no significant difference in survival rate was observed between single cells and shrink-packed cells injected with a 28G needle; both types showed survival rates exceeding 90%. Image 900f shows that quantification determined that single CECs had a survival rate of over 93%, and shrink-packed cells (including cells not integrated into the μ monolayer) had a survival rate of over 97%.

[0080] Shrink-packed μ-monolayers exhibit different growth characteristics compared to single CECs. To evaluate the potential use of shrink-packed μ-monolayers in cell injection therapy, we determined whether they are thought to bind to and proliferate in the exfoliated stroma, primarily collagen I. Both single CECs and shrink-packed μ-monolayers were injected onto compressed collagen I gel, which acts as an exfoliated stroma mimetic. Samples were fixed and stained 6 and 24 hours after injection to observe cell structure and external growth.

[0081] Referring to Figure 10, as shown in image 1000a, six hours after injection, the single CECs were mostly rounded, with very little observed elongation. F-actin staining indicated a lack of filamentous structures in the cell cytoskeleton. In addition, ZO-1 was not observed. In contrast, CECs derived from shrink-packaged μ monolayers maintained their cytoskeletal structure and tight junctions, as is evident from F-actin filaments at the cell boundary and continuous ZO-1 expression (two lower images in 1000a). More specifically, six hours after re-seeding on collagen I gel, the CECs maintained Z0-1 expression and F-actin cytoskeleton while growing from the ECM scaffold. Cells surrounding the shrink-packaged CECs also expressed Z0-1. In contrast, the single CECs did not establish F-actin cytoskeleton or Z0-1 expression.

[0082] Image 1000b shows a comparison of single CECs (top image) and packaged monolayers (bottom image). 3D views of cells 6 hours after seeding show the difference between single CECs and shrink-packaged CECs. Examination of the 3D samples revealed that single CECs were very round and had very little intercellular interaction, while shrink-packaged μ-monolayers inverted with the ECM scaffold present at the center of the cell, which was directly bound to the stromal mimetic (1000b, bottom). After 24 hours, single CECs had covered most of the stromal mimetic and had a more defined cytoskeletal structure compared to the 6-hour CECs, where a lot of F-actin stressed the filaments throughout the cell body. Image 1000c shows that at 24 hours, single CECs had begun to extend and covered almost the entire scaffold. At 24 hours, the CECs had already grown from the ECM scaffold and formed a nearly complete monolayer. In image 1000d, the nucleus is dark (shaded blue), Z0-1 is light (shaded red), COL4 is shaded magenta, and F-actin is shaded green. The scale bar is 50 μm, with the exception of the orthogonal view, where the scale bar is 20 μm. The single CEC showed very slight discontinuous ZO-1 at the cell boundary. In contrast, the shrink-packed μ monolayer had continuous ZO-1 at all cell boundaries and a cortical F-actin cytoskeleton that closely resembled the structure of the in vivo CEC, as shown in image 1000d. The ECM scaffold was still visible after 24 hours, as indicated by the arrow in image 1000d (right image).

[0083] Shrink-packaged μ monolayers are integrated into CE monolayers to increase density in vivo. Many patients require corneal transplants due to impairments caused by low cell density rather than disease, and therefore can benefit from cell injections to boost cell density without full transplantation. Thus, we implement the integration of shrink-packaged CECs into existing CE monolayers. Low-density CE monolayers were formed by seeding late-passed BCECs onto collagen I gel stromal mimics used in previous experiments. Shrink-packaged μ monolayers or an equivalent number of single CECs were then injected into confluent CE monolayers using a 300 needle. CECs used for both single-cell and μ monolayer injections were labeled with CellTracker Green, and the cells after injection and a CE monolayer that was not injected with any cells served as a control. Samples were fixed, stained, and analyzed 3, 7, and 14 days after injection. Images of each of these data points are shown in Figure 11A on grid 1100a. At each time point, very few single CECs may be integrated into the existing monolayer. On day 3, the integrated shrink-packed μ monolayers appeared very densely packed compared to cells derived from the CE monolayer, and still possessed some 3D structure. By day 7, the shrink-packed μ monolayers were fully integrated within the monolayer, although CellTracker-positive cells derived from the μ monolayer appeared smaller than those derived from the existing monolayer. Image 400a is labeled to show control cells, single cells, and packaging cells. On day 14, the shrink-packed μ monolayers appeared here to be comparable in size to cells derived from the existing monolayer, indicating that monolayer integration and density equilibrium had been achieved. CellTracker-labeled single cells and shrink-packed cells were visible at each time point. However, a considerable number of shrink-packed cells were present at each time point, and the ECM scaffold was still visible even 14 days after injection.

[0084] The confocal images of the integrated shrink-wrapped μ monolayers at each point in time were quantified using heatmap representation. The average pixel density of the CellTracker signal for each integrated μ monolayer was calculated and the results are displayed in the heatmap shown in image 1100b. These heatmaps confirm the observation that the shrink-wrapped CEC μ monolayers first integrate into densely packed clusters that expand over time. The heatmap of the areas occupied by CellTracker-labeled shrink-wrapped cells shows that over 14 days, the cells first integrate into tightly packed clusters, and then the density begins to equilibrium as the cells gradually expand. For example, the number is 33 on day 3, 37 on day 7, and 40 on day 14.

[0085] To quantify whether shrink-wrapped μ monolayers increased the cell density of CE monolayers, confocal images were obtained, and cell density was determined by counting nuclei per image and dividing by the image area of ​​each sample type at each time point. Ten images were counted per sample to obtain the average per sample, and the average per sample type was calculated from either three or four individual samples. The results are shown in Graph 1100c. On day 3, CE monolayers with integrated shrink-wrapped μ monolayers (1731±267 cells / mm2) had a significantly higher cell density than the control (1016±75 cells / mm2), but there was no significant difference compared to CE monolayers with integrated single CECs (1253±31 cells / mm2). On day 7, the CE monolayer with integrated shrink-wrapped μ monolayers (1631±58 cells / mm2) had a significantly higher density than both the control (989±11 cells / mm2) and the monolayer with integrated single CECs (1220±56 cells / mm2). Similarly, on day 14, the CE monolayer with integrated shrink-wrapped μ monolayers (1545±95 cells / mm2) had a significantly higher density than both the control (994±104 cells / mm2) and the monolayer with integrated single CECs (1224±66 cells / mm2). The large standard deviation in cell density observed on day 3 in the shrink-wrapped samples decreased on days 7 and 14, further supporting observations quantitatively derived from confocal images and heatmaps. This continued integration and equilibration also explains the decrease in cell density observed from day 3 to day 14, where the overall cell density does not necessarily decrease, but rather becomes more uniform across the entire CE monolayer.

[0086] While these long-term results are useful, it was also important to understand the short-term integration of shrink-packaged CECs to determine whether the method clinically used for single-cell injections, where the patient's face remained face down for 3 hours, is considered sufficient for the injection of shrink-packaged μ monolayers. Image 1100d in Figure 11B shows a time-lapse image derived from a live confocal image of the integration of shrink-packaged CECs into an existing monolayer. At 3 hours, the cells began to bind and integrate, and by 43 hours, the cells were almost completely integrated into the monolayer. Live confocal images of shrink-packaged μ monolayer integration were acquired by labeling shrink-packaged cells with CellTracker green and cells in the CE monolayer with CellTracker orange. Z-stacks were collected hourly for 48 hours, and time-lapse video is viewed in auxiliary video S1. Three hours after injection, the shrink-packaged μ monolayer began to bind and flattened at the top of the CE monolayer. CE monolayer cells continued to migrate over 48 hours, integrating the shrink-packaged μ monolayer. Up to 43 hours, the shrinkable μ monolayer is almost completely integrated into the CE monolayer. This video also confirms previous results showing that cells in the shrinkable μ monolayer are packed much more densely than CE monolayer cells at an early stage. The shrinkable μ monolayer begins integration into ex vivo rabbit cornea within 3 hours. In vitro live image results suggest that 3 hours of the patient lying face down is considered sufficient time for the shrinkable μ monolayer to integrate after injection, although the in vitro CE monolayer potentially has a higher ability to migrate and take up injected cells compared to in vivo CE.

[0087] As the next step to confirm these results before moving on to an in vivo study, shrink-wrapped rabbit μ-monolayers were injected into the anterior chamber of ex vivo rabbits, the eyes were incubated with the cornea facing downwards for 3 hours, and then the eyeballs were immobilized. After immobilization, the corneas were excised, rinsed vigorously three times to remove all unbound cells, and stained for Z0-1, F-actin, and nuclei. Confocal images of the cornea showed numerous shrink-wrapped μ-monolayers bound together throughout the cornea. The shrink-wrapped μ-monolayers were still round and clustered together with the ECM scaffold in the center, as shown in image 1200 of Figure 12. The confocal images showed that the shrink-wrapped cells had begun to integrate with the ex vivo CE, and the ECM scaffold was observed between the shrink-wrapped cells and the existing monolayers. The vertical and horizontal lines indicate the locations where the orthogonal views were obtained.

[0088] Figure 13 shows the results of shrink-packed μ-monolayers integrated into healthy rabbit CE cells in vivo. To test the feasibility of using shrink-packed μ-monolayers to increase patient CE cell density, a study was conducted using the eyes of three healthy New Zealand white rabbits. DiO-labeled shrink-packed μ-monolayers were injected into the anterior chamber of one eye of each rabbit. The rabbits were placed with the injected eye side down for 3 hours to allow the shrink-packed μ-monolayers to integrate. The rabbits were then observed for 7 days before being sacrificed and enucleated. On day 7, the injected eyes of all three rabbits remained clear, and there were no external signs of inflammation or swelling. Image 1300a is a photograph of the rabbit eye on day 7, showing that the cornea remained healthy and clear. After enucleation, the eyes were immobilized in the anterior chamber via injection of 2% paraformaldehyde (PBS++), and the eyeballs were immersed in 2% paraformaldehyde (PBS++) for 24 hours, followed by staining for Z0-1 and tight junctions via the nuclei. Confocal microscopy images showed that DiO-labeled cells were present in the injected eyes of all three rabbits, and wide-area tile scans showed numerous clusters of shrink-packed μ monolayers integrated throughout the cornea. Image 1300b shows a wide-area tile-scanned confocal image showing that 7 days after injection, many areas with DiO (green)-labeled shrink-packed cells were still present in the cornea. Higher magnification images showed that the shrink-packed μ monolayer cells tightly integrated with healthy rabbit CE, and Z0-1 was continuously present at all cellular boundaries between the DiO-labeled cells and the natural rabbit CEC. Image 1300c shows a magnified view of the area in 1300b, highlighted by a square, showing that DiO-labeled shrink-packaging cells are integrated into healthy rabbit CE, exhibiting continuous tight junctions (Z0-1) between the shrink-packaging CECs and natural CECs. The ECM scaffold is also still visible, as indicated by the arrow, and the shrink-packaging CECs are in the same plane as the cell-free monolayer on top of the CE, showing uninterrupted integration. The ECM scaffold remains visible, providing further evidence that the shrink-packaging μ monolayer has integrated and become part of the in vivo rabbit CE.

[0089] Figure 14 shows images 1400a–c and graph 1400d illustrating the integration of shrink-packaged CECs into healthy rabbit corneal endothelium, increasing their local density. In image 1400a, very few integrated single cells are present in the healthy rabbit endothelium. In contrast, in image 1400b, many shrink-packaged μ monolayers are integrated into the healthy rabbit endothelium. Image 1400c shows a close-up image of the integrated μ monolayer. The ECM is still central but beneath the cell bodies of the injected cells derived from the fully integrated μ monolayer. Graph 1400d shows the increased local cell density 7 days post-injection in the area where the μ monolayer was integrated, compared to other areas of the cornea in the same field of view.

[0090] Figure 15 shows a method 1500 for encapsulating cells. A tissue scaffold stamp is coated with an extracellular matrix compound (1510). The tissue scaffold stamp is placed on a thermoresponsive substrate (1520). Cell cultures are seeded onto the tissue scaffold stamp (1530). The cell cultures are incubated on the tissue scaffold stamp at a specified temperature (1540). The cell cultures form a monolayer on the tissue scaffold stamp (1550), with the monolayer boundaries configured to maintain the expression of intercellular junctions, and the intercellular junctions of the monolayer express tension. The thermoresponsive substrate is dissolved by decreasing the temperature. The tissue scaffold stamp is removed from the cell patch (1560), and a microtissue structure is formed by dissolving the tissue scaffold stamp in a solvent. The microtissue structure is folded while suspended in the solvent (1570), allowing the cell patch to fold the extracellular matrix compound, and the folding of the matrix compound also causes the microtissue structure to fold. The folded microtissue structure was recovered from the solvent using centrifugation (1580). The folded microtissue structure was then administered to a living organism (1590).

[0091] Numerous exemplary embodiments have been described. Nevertheless, it will be understood by those skilled in the art that various modifications can be made without departing from the spirit and scope of the art described herein.

Claims

1. A method for encapsulating cells in microtissue, Coating tissue scaffold stamps with extracellular matrix compounds, The tissue scaffold stamp is placed on a thermoresponsive substrate, The process involves seeding cell cultures onto the aforementioned tissue scaffold stamp, The incubation of the cell culture on the tissue scaffold stamp at a specified temperature, wherein the cell culture forms a cell patch that binds to the extracellular matrix compound. The cell patch forms a monolayer on the tissue scaffold stamp, and the boundary of the monolayer maintains the expression of intercellular junctions. Removing the aforementioned thermally responsive substrate, The tissue scaffold stamp is removed from the cell patch to form a microtissue structure around the cell patch, The microstructure is folded by suspending the microstructure in a solvent, The folded microstructure is recovered from the solvent, The aforementioned folded microtissue structure is administered to a living organism. Methods that include...

2. The aforementioned tissue scaffold is formed into a tubular structure, Forming cell patches including tubular shapes based on the tubular structure of the aforementioned tissue scaffold. The method according to claim 1, further comprising:

3. The method according to claim 2, wherein the cell patch includes a fragment of a blood vessel.

4. The method according to claim 1, further comprising adding an antibody to the cell patch.

5. The method according to claim 1, wherein administering a microtissue structure to a living organism includes injecting the microtissue structure.

6. The cell patch comprises corneal endothelial cells, and the method is Introducing the aforementioned cell patch into the cornea, Using gravity to ensure contact between the cell patch and the cornea The method according to claim 5, further comprising:

7. The method according to claim 5, wherein the size of the microstructure is proportional to the size of the tissue scaffold stamp, and the size of the microstructure is a part of the diameter of the injection device.

8. The method according to claim 1, wherein the tissue scaffold stamp contains an organosilicon compound.

9. The method according to claim 8, wherein the organosilicon compound comprises polydimethylsiloxane.

10. The method according to claim 1, wherein the extracellular matrix compound comprises a protein containing one or more of collagen IV, laminin, fibroblast growth factor protein, and vascular endothelial growth factor protein.

11. The method according to claim 1, wherein the arrangement of the tissue scaffold stamps includes printing the tissue scaffold stamps onto the thermoresponsive substrate.

12. The method according to claim 1, wherein the thermally responsive substrate comprises a PIPAAm polymer.

13. The method according to claim 1, wherein the tissue scaffold stamp forms a regular shape.

14. The aforementioned tissue scaffold stamp is 250 μm ２ The method according to claim 1, comprising surface dimensions less than or approximately equal to thereto.

15. The method according to claim 1, wherein the cell patch contains 10 to 100 cells.

16. A cell patch containing a cell monolayer that maintains the expression of intercellular junctions and the cytoskeleton of cells within the cell patch, A microtissue structure folded around the cell patch, comprising an extracellular matrix configured to provide a physical barrier between the cell patch and the external environment. A system that includes this.

17. The system according to claim 16, wherein the extracellular matrix comprises a protein including one or more of collagen IV, laminin, fibroblast growth factor protein, and vascular endothelial growth factor protein.

18. The system according to claim 16, wherein the microstructure forms a tubular structure.

19. The system according to claim 16, wherein the monolayer contains 10 to 100 cells.

20. The system according to claim 16, wherein the cell patch includes muscle tissue, and growth factors are added to the extracellular matrix to promote intravascular growth of the muscle tissue.

Images