# US 11,078,454 B2 · Bioreactor and method of forming complex three-dimensional tissue constructs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,454 B2 - Document ID: US11078454B2 - Status: Granted - Kind: B2 - Application number: 16/197,944 - Filed: 2018-11-21 - Published: 2019-05-23 - Granted: 2021-08-03 - Earliest claimed: 2015-07-15 - Assignees: The Regents of the University of Michigan - Applicants: The Regents of the University of Michigan - Inventors: Lisa M. Larkin; Ellen M. Arruda; Michael J. Smietana; Pablo Moncada-Larrotiz - Web page: https://patentgrep.com/patent/US11078454B2 ## CPC - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - A61F 2/08: Muscles; Tendons; Ligaments - C12M 23/04: {Flat or tray type, drawers (C12M23/10, C12M23/12, C12M23/16 take precedence)} - C12M 23/08: {Flask, bottle or test tube} - C12M 23/34: {Internal compartments or partitions} - C12M 23/50: {Means for positioning or orientating the apparatus (C12M41/08 takes precedence)} - C12M 29/10: {Perfusion} - C12M 35/08: {Chemical, biochemical or biological means, e.g. plasma jet, co-culture} - A61F 2240/001: Designing or manufacturing processes - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C) ## Abstract A bioreactor and method of forming complex three-dimensional tissue constructs in a single culture chamber. The bioreactor and methods may be used to form multi-phasic tissue constructs having tissue formed from multiple cell types in a single culture chamber. The bioreactor includes at least one translation mechanism to facilitate translation of one or more tissue constructs without direct user intervention, thereby providing a closed, sterile environment for complex tissue fabrication. The bioreactor may be used as a stand-alone device or as part of a large-scale system including many bioreactors. The large-scale system may include a perfusion system to monitor and regulate the tissue culture environment. ## Claims ### Claim 1 A method of forming a multi-phasic tissue construct in a single culture chamber, comprising the steps of: forming a first tissue construct in the single culture chamber; translating the first tissue construct from a first substrate toward a second substrate via at least one translation mechanism that at least partially extends between the first substrate and the second substrate; adding cells to the single culture chamber containing the first tissue construct; and culturing the cells to form a second tissue construct in the single culture chamber, wherein the first tissue construct and the second tissue construct are formed from different cell types and together comprise the multi-phasic tissue construct. ### Claim 2 (depends on 1) The method of claim 1, wherein the cells are allogeneic mesenchymal stem cells (MSCs). ### Claim 3 (depends on 1) The method of claim 1, wherein the multi-phasic tissue construct includes muscle tissue, tendon tissue, nerve tissue, or some combination of one or more muscle tissue, tendon tissue, and nerve tissue. ### Claim 4 (depends on 1) The method of claim 1, wherein the first tissue construct is a bone tissue construct, the second tissue construct is a ligament tissue construct, and the multi-phasic tissue construct is a bone-ligament tissue construct. ### Claim 5 (depends on 4) The method of claim 4, wherein the culture chamber is at least partially divided into two culture vessels, and two bone tissue constructs are formed in each culture vessel of the culture chamber, and the ligament tissue construct extends between the two bone tissue constructs so that the multi-phasic tissue construct is a bone-ligament-bone (BLB) tissue construct. ### Claim 6 (depends on 1) The method of claim 1, wherein the step of adding cells takes place before the cells form a confluent tissue monolayer. ### Claim 7 (depends on 1) The method of claim 1, wherein the step of culturing the cells includes forming a spontaneously delaminating tissue monolayer. ### Claim 8 (depends on 1) The method of claim 1, wherein the first substrate and the second substrate comprise two walls of the culture chamber. ### Claim 9 (depends on 1) The method of claim 1, wherein the first tissue construct shrinks while translating away from the first substrate. ### Claim 10 (depends on 9) The method of claim 9, further comprising the step of translating the second tissue construct from the second substrate toward the first substrate via at least one translation mechanism that at least partially extends between the first substrate and the second substrate, wherein the second tissue construct shrinks while translating away from the second substrate. ### Claim 11 A method of forming a complex three-dimensional tissue construct in a single culture chamber, comprising the steps of: adding cells from a first cell source to a first substrate in the culture chamber; adding cells from a second cell source to a second substrate in the culture chamber; culturing the first cell source and the second cell source to form a first tissue construct and a second tissue construct; translating the first and second tissue constructs via one or more translation mechanisms to a co-culture zone between the first and second substrates; and forming the complex three-dimensional tissue construct having the second tissue construct of the second cell source cultured with the first tissue construct of the first cell source. ### Claim 12 (depends on 11) The method of claim 11, wherein the culture chamber includes a body for supporting the first substrate and the second substrate. ### Claim 13 (depends on 12) The method of claim 12, wherein the body is a polygonal-shaped body. ### Claim 14 (depends on 12) The method of claim 12, further comprising the step of removing the first substrate and the second substrate from the body of the culture chamber. ### Claim 15 (depends on 13) The method of claim 13, further comprising the step of reusing the body with new first and second substrates to form another complex three-dimensional tissue construct. ### Claim 16 (depends on 11) The method of claim 11, further comprising the step of using the culture chamber as a container for preservation, storage, and shipping of the complex three-dimensional tissue construct. ### Claim 17 (depends on 11) The method of claim 11, further comprising the step of adding cells from a third cell source to a third substrate in the culture chamber and culturing the cells of the third cell source with the first cell source and the second cell source to form the first tissue construct, the second tissue construct, and a third tissue construct, wherein the translating step includes translating the third tissue construct via the one or more translation mechanisms to the co-culture zone that is between the first, second, and third substrates, and forming the complex three-dimensional tissue construct having the third tissue construct of the third cell source cultured with the first and second tissue constructs of the first and second cell sources. ### Claim 18 (depends on 17) The method of claim 17, wherein the culture chamber includes a trigonal-shaped body for supporting the first substrate, the second substrate, and the third substrate.