# US 11,898,159 B2 · Methods of making spheroids including biologically-relevant materials

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,898,159 B2
- Document ID: US11898159B2
- Status: Granted
- Kind: B2
- Application number: 17/125,322
- Filed: 2020-12-17
- Published: 2021-06-10
- Granted: 2024-02-13
- Earliest claimed: 2013-08-21
- Assignees: University of Louisville Research Foundation, Inc.
- Applicants: University of Louisville Research Foundation, Inc.
- Inventors: Stuart K. Williams; Jeremy S. Touroo; James B. Hoying
- Web page: https://patentgrep.com/patent/US11898159B2

## CPC

- C12N 5/0012: {Cell encapsulation}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)}
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0075: {using microcarriers}
- B33Y 10/00: Processes of additive manufacturing
- B33Y 80/00: Products made by additive manufacturing
- C12N 2513/00: 3D culture
- C12N 2533/74: Alginate
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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}
- 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

Methods of making a spheroid are provided whereby a suspension is first produced including one or more biologically-relevant materials dispersed within a biocompatible medium. A droplet of the suspension is then bioprinted into a salt solution by bringing the droplet into contact with a surface of the salt solution in a controlled manner to reproducibly yield a spheroid containing a desired size and a desired amount of biologically-relevant materials.

## Claims

### Claim 1

A pre-vascularized spheroid comprising one or more microvascular fragments surrounding a composition that comprises one or more biologically-relevant materials, said biologically-relevant materials and microvascular fragments being dispersed in a hydrogel comprising alginate, said spheroid having uniform dimensions and a diameter between 500 and 2,500 microns, wherein said spheroid is produced by a method comprising:
providing a first suspension comprising one or more biologically-relevant materials dispersed within a biocompatible medium, wherein the biocompatible medium is a hydrogel comprising alginate;
providing a second suspension comprising one or more microvascular fragments dispersed within a biocompatible medium, wherein the biocompatible medium is a hydrogel comprising alginate;
providing a bioprinter having a first delivery pen surrounded by a second delivery pen;
placing the first suspension in the first delivery pen and the second suspension in the second delivery pen; and
extruding the first suspension from the first delivery pen and the second suspension from the second delivery pen in a substantially simultaneous manner such that a droplet is formed with the second suspension encapsulating the first suspension, thereby forming the pre-vascularized spheroid;
wherein the biologically-relevant materials comprise stromal vascular fraction cells or stem cells.

### Claim 2 (depends on 1)

The pre-vascularized spheroid of claim 1, wherein the method further comprises a step of placing the droplet against a surface of a salt solution.

### Claim 3 (depends on 2)

The pre-vascularized spheroid of claim 2, wherein the salt solution comprises CaCl2).

### Claim 4 (depends on 2)

The pre-vascularized spheroid of claim 2, wherein the step of bringing the droplet into contact with the surface of the salt solution is performed on a direct-write bioprinter.

### Claim 5

A pre-vascularized spheroid comprising one or more microvascular fragments surrounding a composition that comprises one or more biologically-relevant materials, said biologically-relevant materials and microvascular fragments being dispersed in a hydrogel comprising alginate, said spheroid having uniform dimensions and a diameter between 500 and 2,500 microns, wherein said spheroid is produced by a method comprising:
providing a droplet of a suspension on a delivery pen tip, wherein the suspension comprises one or more biologically-relevant materials dispersed within a biocompatible medium, wherein the biocompatible medium is a hydrogel comprising alginate;
bringing the droplet into contact with a surface of a salt solution by bringing the delivery pen tip toward the salt solution; and
compressing the droplet against the surface of the salt solution for a period of time until the spheroid is formed;
wherein the biologically-relevant materials comprise stromal vascular fraction cells or stem cells.

### Claim 6 (depends on 5)

The pre-vascularized spheroid of claim 5, wherein the salt solution comprises CaCl2).

### Claim 7 (depends on 5)

The pre-vascularized spheroid of claim 5, wherein the delivery pen tip is not in contact with the salt solution when the spheroid is formed.

### Claim 8 (depends on 1)

The pre-vascularized spheroid of claim 1, wherein the spheroid has a diameter between 1500 and 2,500 microns.

### Claim 9 (depends on 1)

The pre-vascularized spheroid of claim 1, wherein the spheroid has a diameter between 800 and 1700 microns.

### Claim 10 (depends on 5)

The pre-vascularized spheroid of claim 5, wherein the spheroid has a diameter between 1500 and 2,500 microns.

### Claim 11 (depends on 5)

The pre-vascularized spheroid of claim 5, wherein the spheroid has a diameter between 800 and 1700 microns.