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JP-7854716-B2 - Monolayer cell patch in an extracellular matrix scaffold

JP7854716B2JP 7854716 B2JP7854716 B2JP 7854716B2JP-7854716-B2

Inventors

  • ファインバーグ，アダム ウォルター
  • シムコ，レイチェル

Assignees

  • カーネギー－メロン ユニバーシティ

Dates

Publication Date
20260507
Application Date
20201019
Priority Date
20191018

Claims (14)

  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 cell boundaries in the monolayer, formed by ZO-1 at the cell continuity boundary before injection and the cortical F-actin cytoskeleton on the cell patch, maintain the expression of intercellular junctions, and the injected monolayer forms a cell density of 1250 to 1700 cells/square millimeter. 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, To recover the aforementioned folded microstructure from the solvent, Includes, A method wherein the cell patch and tissue scaffold stamp forming the monolayer are injectable into tissue, and the cell patch is released from the microtissue structure and integrated into the tissue to form a cell density of 1250 to 1700 cells/square millimeter in the tissue.
  2. The aforementioned tissue scaffold is formed into a tubular structure, The method according to claim 1, further comprising forming a single layer including a tubular shape based on the tubular structure of the tissue scaffold.
  3. The method according to claim 2, wherein the monolayer 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 the cell patch comprises corneal endothelial cells.
  6. The method according to claim 1 , 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.
  7. The method according to claim 1, wherein the tissue scaffold stamp contains an organosilicon compound.
  8. The method according to claim 7 , wherein the organosilicon compound comprises polydimethylsiloxane.
  9. 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.
  10. The method according to claim 1, wherein the arrangement of the tissue scaffold stamps includes printing the tissue scaffold stamps onto the thermoresponsive substrate.
  11. The method according to claim 1, wherein the thermally responsive substrate comprises a PIPAAm polymer.
  12. The method according to claim 1, wherein the tissue scaffold stamp forms a regular shape.
  13. The method according to claim 1, wherein the tissue scaffold stamp has a surface dimension of less than or approximately equal to 250 μm² .
  14. The method according to claim 1, wherein the cell patch contains 10 to 100 cells.

Description

Priority Claim 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. Government Rights: This invention was made with government support under authorization number 1RO1EY024642-01A1 granted by the National Institutes of Health. The Federal Government has certain rights in this invention. This disclosure relates to the formation of microtissues and the application of microtissues to living organisms. 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 repaired 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 cellular structure and phenotype. In addition, injected single cells often die immediately after injection due to a combination of stress from the physical injection process and insufficient binding to the desired tissue. 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, enabling them to repair damaged tissue after delivery. 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). The extracellular microenvironment (ECM) can include an inclusion material that replicates the desired microenvironment for a cell patch in vivo. For example, the ECM can be constructed to have a density, structure, and/or composition similar to that of the natural ECM, and these cells are surrounded in vivo. The ECM provides a unique microenvironment that more closely matches that present in vivo, thus improving the ability to modulate cell behavior. Cell patches can contain a cell monolayer. The monolayer is formed to include cell expression 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. Shrink-packaged monolayers offer one or more of the following advantages: In contrast to enzyme-based methods that release cells from a scaffold, the biological characteristics of the cells are maintained for in vivo administration. 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. For example, when introduced in vivo in various situations (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. As 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. As will be discussed later, monolayers offer other advantages over single-cell techniques. For example, endothelium and/or epithelium act as barriers for cell administration. Since the administered cell monolayer is a foam cell patch, the endothelium allows the patch to be incorporated. 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 one patch to another within the 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. In some embodiments, cell-additive materials are added to the extracellular matrix to promote patterning. For example, antibodies can be added for delivery 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., cyt