# US 2018/0002658 A1 · Hypothermic 3d bioprinting of living tissues supported by perfusable vasculature

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- Number: US 2018/0002658 A1
- Document ID: US20180002658A1
- Status: Published application
- Kind: A1
- Application number: 15/709,392
- Filed: 2017-09-19
- Published: 2018-01-04
- Earliest claimed: 2015-03-20
- Assignees: William Marsh Rice University
- Applicants: William Marsh Rice University
- Inventors: Jordan Miller; Anderson Ta; Bagrat Grigoryan
- Web page: https://patentgrep.com/patent/US20180002658A1

## CPC

- C12N 5/0062: {General methods for three-dimensional culture}
- B29C 64/129: characterised by the energy source therefor, e.g. by global irradiation combined with a mask
- B29C 64/393: for controlling or regulating additive manufacturing processes
- B33Y 10/00: Processes of additive manufacturing
- B33Y 50/02: for controlling or regulating additive manufacturing processes
- B33Y 70/00: Materials specially adapted for additive manufacturing
- C12M 3/00: Tissue, human, animal or plant cell, or virus culture apparatus
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0641: {Erythrocytes}
- B29K 2071/02: {Polyalkylene oxides, e.g. PEO, i.e. polyethylene oxide, or derivatives thereof}
- C12N 2513/00: 3D culture
- C12N 2533/30: Synthetic polymers
- C12N 2537/00: Supports and/or coatings for cell culture characterised by physical or chemical treatment
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B29C: SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING (making preforms B29B11/00; making laminated products by combining previously unconnected layers which become one product whose layers will remain together B32B37/00 - B32B41/00)
- B33Y: ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- 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}
- B29K: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES B29B, B29C OR B29D, RELATING TO MOULDING MATERIALS OR TO MATERIALS FOR {MOULDS, } REINFORCEMENTS, FILLERS OR PREFORMED PARTS, e.g. INSERTS

## Abstract

The present disclosure provides compositions and methods for producing hydrogel matrix constructs. Methods of using hydrogel matrix constructs for tissue repair and regeneration and for the oxygenation of red blood cells are also disclosed.

## Claims

### Claim 1

A prepolymerization solution comprising: a photosensitive polymer having a molecular weight of greater than 2,000 Daltons and comprising at least two vinyl groups per molecule of polymer; a photoinitiator; and a biocompatible, light-absorbing additive material suitable to control light penetration.

### Claim 2 (depends on 1)

The prepolymerization solution of claim 1 further comprising a cell.

### Claim 3 (depends on 1)

The prepolymerization solution of claim 1, wherein the biocompatible, light-absorbing additive material is organic.

### Claim 4 (depends on 1)

The prepolymerization solution of claim 1, wherein the biocompatible, light-absorbing additive material is tartrazine or curcumin.

### Claim 5 (depends on 1)

The prepolymerization solution of claim 1 comprising a water content of 10 wt % to about 99.5 wt %.

### Claim 6 (depends on 1)

The prepolymerization solution of claim 1, wherein the polymer is poly(ethylene glycol) and wherein the at least two vinyl groups are methacrylate or acrylamide.

### Claim 7 (depends on 1)

The prepolymerization solution of claim 1 wherein the photo-initiator is lithium acylphosphinate.

### Claim 8

A composition comprising:
a hydrogel matrix comprising a plurality of layers, each layer comprising a cross-linked polymer network formed from a photosensitive polymer having a molecular weight greater than 2,000 Daltons, wherein the hydrogel matrix has a elastic modulus of from about 1 kilopascal to about 200 kilopascals;
a first elongated void in the hydrogel matrix providing a first tubular channel;
a second elongated void in the hydrogel matrix providing a second tubular channel;
wherein the first tubular channel and the second tubular channel are perfusable;
wherein the first tubular channel does not intersect the second tubular channel; and
wherein the second tubular channel interpenetrates the first tubular channel.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the first tubular channel or second tubular channel is lined with cells.

### Claim 10 (depends on 8)

The composition of claim 8, wherein the first and second tubular channel each comprise a horizontal segment, wherein a cross-section in the horizontal segment intersects more than one layer of the bulk hydrogel matrix.

### Claim 11 (depends on 8)

The composition of claim 8, wherein the hydrogel matrix is porous.

### Claim 12 (depends on 8)

The composition of claim 8, wherein the first tubular channel is filled with a fluid selected from the group consisting of culture media, blood, urine, and bile.

### Claim 13 (depends on 8)

The composition of claim 8, wherein the second tubular channel is filled with oxygen.

### Claim 14 (depends on 8)

The composition of claim 8, wherein the first tubular channel and second tubular channel have a diameter of 500 microns or less.

### Claim 15 (depends on 8)

The composition of claim 8, wherein each layer of the hydrogel matrix has a thickness of 100 microns or less.

### Claim 16 (depends on 8)

The composition of claim 8, wherein the first tubular channel and second tubular channel are expandable in response to increases in pressure therein.

### Claim 17 (depends on 8)

The composition of claim 8, wherein one or more layers of the hydrogel matrix has cells embedded therein.

### Claim 18

A process for manufacturing a multi-layer hydrogel matrix construct, comprising:
a) creating a 3D model of the multi-layer hydrogel matrix construct using a design software, wherein the 3D model of the multi-layer hydrogel matrix construct comprises a first computational algorithm that yields a first elongated void in the multi-layer hydrogel matrix construct providing a first tubular channel, and a second computational algorithm that yields a second elongated void in the multi-layer hydrogel matrix construct providing a second tubular channel, wherein the second computational algorithm results in the second tubular channel interpenetrating the first tubular channel;
b) converting the 3D model to a format suitable for use in a 3D printing software to yield a formatted 3D model;
c) importing the formatted 3D model into the 3D printing software, wherein the 3D printing software is programmed to slice the formatted 3D model into multiple 2D xy images;
d) supplying a prepolymerization solution to a vat associated with a build platform of a 3D printer, wherein the vat is transparent, and wherein the prepolymerization solution comprises a photosensitive polymer having a molecular weight of greater than 2,000 Daltons and at least two vinyl groups per molecule of polymer, a light-absorbing additive material to control light penetration, and a photoinitiator;
e) positioning a mobile Z-axis stage of the 3D printer at a distance from the vat, wherein the Z-axis stage includes a surface sufficient for gelled material to adhere thereto, wherein the distance between the surface and an inner bottom surface of the vat is equivalent to a desired layer thickness of the multi-layer hydrogel matrix construct;
f) projecting a pattern being onto the inner bottom surface of the vat, wherein the pattern corresponds to one of the multiple 2D xy images;
g) polymerizing a layer of the multi-layer hydrogel matrix construct consistent with the pattern; and
h) repeating steps d through g one or more times, wherein the mobile Z-axis stage is moved so that the distance moved is equivalent to the desired thickness of each subsequent layer, and wherein the same or a different pattern is displayed for each subsequent layer.

### Claim 19 (depends on 18)

The process of claim 18, wherein the first computational algorithm is a Hilbert Curve, derived from knot theory, or conforms to Murray's law.

### Claim 20 (depends on 18)

The process of claim 18, wherein the prepolymerization solution further comprises one or more cells.