# US 2020/0399594 A1 · Morphogenic compound-releasing microspheres and use in bioink

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- Number: US 2020/0399594 A1
- Document ID: US20200399594A1
- Status: Published application
- Kind: A1
- Application number: 16/971,634
- Filed: 2019-02-22
- Published: 2020-12-24
- Earliest claimed: 2018-02-22
- Assignees: UVic Industry Partnerships Inc.
- Applicants: UVic Industry Partnerships Inc.
- Inventors: Andrew Agbay; Laura De La Vega; Stephanie Michelle Willerth
- Web page: https://patentgrep.com/patent/US20200399594A1

## CPC

- A61L 27/383: {Nerve cells, e.g. dendritic cells, Schwann cells}
- C12N 5/0619: {Neurons}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- B33Y 10/00: Processes of additive manufacturing
- B33Y 70/10: Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0623: {Stem cells}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/00: Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N 2513/00: 3D culture
- C12N 2531/00: Microcarriers
- C12N 2533/40: Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
- C12N 2533/72: Chitin, chitosan
- C12N 2537/10: Cross-linking
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The bioink disclosed herein includes one or more cells, a carrier material, and microspheres. The microspheres can include one or more biodegradable polymers and one or more compounds, such as a morphogenic compound. The methods disclosed herein can include three-dimensional bioprinting. Additional methods disclosed herein include producing functional tissue.

## Claims

### Claim 1

A bioink, comprising:
one or more cells;
a carrier material; and
discrete microspheres, the discrete microspheres comprising:
one or more biodegradable polymers; and
one or more compounds,
wherein the one or more compounds release with degradation of the one or more biodegradable polymers.

### Claim 2 (depends on 1)

The bioink of claim 1, wherein one or more cells comprise cell aggregates.

### Claim 3 (depends on 1)

The bioink of claim 1, wherein one or more cells comprise stem cells or progenitor cells.

### Claim at position 4

-5. (canceled)

### Claim 6 (depends on 3)

The bioink of claim 3, wherein the progenitor cells comprise neural progenitor cells.

### Claim 7 (depends on 1)

The bioink of claim 1, wherein the one or more compounds comprise morphogenic compounds.

### Claim 8

(canceled)

### Claim 9 (depends on 7)

The bioink of claim 7, wherein the morphogenic compounds comprise purmorphamine (puro), guggulsterone (GS), or both.

### Claim 10 (depends on 7)

The bioink of claim 7, wherein the morphogenic compounds comprise one or more neurotrophic factors (NTFs), insulin-like growth factor 1 (IGF-1), or any combination thereof.

### Claim 11 (depends on 10)

The bioink of claim 10, wherein the NTFs comprise glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), or both.

### Claim 12 (depends on 1)

The bioink of claim 1, wherein the one or more biodegradable polymers comprise polylactic acid (PLA), polyglycolic acid (PLGA), polycaprolactone (PCL), or any combination thereof.

### Claim 13 (depends on 1)

The bioink of claim 1, wherein the carrier material comprises one or more polymers, crosslinkers, hydrogels, or any combination thereof.

### Claim 14

(canceled)

### Claim 15 (depends on 13)

The bioink of claim 13, wherein the hydrogels comprise methacrylamide chitosan (MAC), fibrin, alginate, or any combination thereof.

### Claim 16 (depends on 1)

A method of three-dimensional bioprinting, comprising bioprinting at least one layer of the bioink of claim 1 onto a surface.

### Claim 17 (depends on 1)

A method of producing functional tissue, comprising:
bioprinting at least one layer of the bioink of claim 1;
measuring one or more markers of differentiation or maturation; and
detecting a level of expression of one or more markers of differentiation or maturation expected in a functional tissue.

### Claim 18

(canceled)

### Claim 19 (depends on 17)

The method of claim 17, wherein the functional tissue is neural tissue.

### Claim 20 (depends on 17)

The method of claim 17, wherein the one or more markers comprise nestin, beta-tubulin III, stage-specific embryonic antigen-4 (SSEA-4), NeuN, oligodendrocyte transcription factor (OLIG2), homeobox HB9 (HB9), paired box protein (PAX6), synapsin I, SRY-box 2 (SOX-2), insulin gene enhancer protein (ISL-1), anti-oligodendrocyte O4 (04), choline acetyltransferase (ChAT), synaptophysin, Oct3/4, c-Myc, kruppel-like factor 4 (KLF4), tyrosine hydroxylase, or neurites.

### Claim 21 (depends on 17)

The method of claim 17, wherein detecting the level of expression of one or more markers of differentiation or maturation expected in a functional tissue comprises detecting:
increased expression of ChAT, O4, NeuN, beta-tubulin III, HB9, synaptophysin, tyrosine hydroxylase, or neurites compared with expression expected in a pluripotent stem cell; or
decreased expression of SSEA-4, Sox-2, Olig2, Isl-1, synapsin I, Oct3/4, c-myc, nestin, PAX6, or KLF4 compared with expression expected in a pluripotent stem cell.

### Claim 22

A method of bioprinting, comprising:
contacting at least one cell type with a microsphere solution and a bioink solution, producing a bioink-cell-microsphere composition;
contacting the bioink-cell composition with a crosslinker, producing a crosslinked bioink-cell composition; and
bioprinting at least one layer of the crosslinked bioink-cell composition using a bioprinter.

### Claim 23 (depends on 22)

The method of claim 22, wherein the bioink solution comprises fibrinogen, alginate, and genipin.

### Claim 24 (depends on 22)

The method of claim 22, wherein the crosslinker comprises CaCl2, chitosan, and thrombin.

### Claim 25 (depends on 22)

The method of claim 22 wherein the at least one cell type forms cell aggregates.

### Claim 26 (depends on 22)

The method of claim 22, wherein the at least one cell type comprises neuronal cells, stem cells, stem cell-derived cells, human induced pluripotent stem cells (hiPSCs), and/or cancer cells.