# US 11,993,785 B2 · Immersion deposition methods and compositions for use in the same

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- Number: US 11,993,785 B2
- Document ID: US11993785B2
- Status: Granted
- Kind: B2
- Application number: 16/533,300
- Filed: 2019-08-06
- Published: 2020-02-13
- Granted: 2024-05-28
- Earliest claimed: 2018-08-07
- Assignees: Wake Forest University Health Sciences
- Applicants: Wake Forest University Health Sciences
- Inventors: Aleksander Skardal; Casey Cuvan Clark
- Web page: https://patentgrep.com/patent/US11993785B2

## CPC

- C12N 5/0062: {General methods for three-dimensional culture}
- G01N 33/5082: {Supracellular entities, e.g. tissue, organisms}
- B33Y 10/00: Processes of additive manufacturing
- B33Y 70/00: Materials specially adapted for additive manufacturing
- B33Y 80/00: Products made by additive manufacturing
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- 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)}
- C12M 33/04: {by injection or suction, e.g. using pipettes, syringes, needles (pipettes in general B01L3/02)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)}
- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- G01N 33/5088: {of vertebrates}
- C12N 2513/00: 3D culture
- C12N 2533/54: Collagen; Gelatin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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}

## Abstract

Provided are methods of preparing organoids and compositions for use in such methods. In some embodiments, a method of depositing a plurality of the cells into a reservoir are provided, the method comprising depositing a gelatin composition into a reservoir; adding a composition comprising a plurality of cells into the gelatin composition, wherein the composition is added into the gelatin composition at a position below the surface of the gelatin composition and at least a portion of the composition and/or plurality of cells are suspended in the gelatin composition; curing at least a portion of the composition comprising the plurality of cells in the gelatin composition; and removing at least a portion of the gelatin composition in the reservoir, thereby depositing the plurality of cells in the reservoir.

## Claims

### Claim 1

A method of depositing a plurality of cells into a reservoir, the method comprising:
depositing a gelatin composition into a reservoir, wherein the gelatin composition is a solution and gelatin is dissolved in the gelatin composition;
adding a composition comprising a plurality of cells into the gelatin composition, wherein the composition is added into the gelatin composition at a position below the surface of the gelatin composition and at least a portion of the composition and/or plurality of cells are suspended in the gelatin composition;
curing at least a portion of the composition comprising the plurality of cells in the gelatin composition; and
removing at least a portion of the gelatin composition in the reservoir, thereby depositing the plurality of cells in the reservoir,
wherein the gelatin composition comprises gelatin at a concentration of about 1 mg/mL to about 15 mg/mL and, at 4° C., the gelatin composition has an elastic modulus of about 5 Pa to about 15 Pa at about 1% to about 100% strain, and
wherein the composition comprising the plurality of cells, at 4° C., has an elastic modulus of about 3 Pa to about 4 Pa at 1%-10% strain and the elastic modulus of the composition comprising the plurality of cells, at 4° C., is within an order of magnitude of the elastic modulus of the gelatin composition.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reservoir is a well in a 96-well plate or 384 well-plate.

### Claim 3 (depends on 1)

The method of claim 1, wherein depositing the gelatin composition into the reservoir comprises depositing about 25 μL to about 350 μL of the gelatin composition into the reservoir.

### Claim 4 (depends on 1)

The method of claim 1, wherein the gelatin composition has a viscosity prior to, during, and/or after deposition of about 0.01 Pascals*seconds (Pa·s) to about 20 Pa·s.

### Claim 5 (depends on 1)

The method of claim 1, wherein adding the composition comprising the plurality of cells into the gelatin composition comprises extruding the composition into the gelatin composition using a syringe and/or printer.

### Claim 6 (depends on 1)

The method of claim 1, wherein curing at least a portion of the composition comprising the plurality of cells comprises exposing at least a portion of the composition comprising the plurality of cells to light.

### Claim 7 (depends on 1)

The method of claim 1, wherein removing at least a portion of the gelatin composition comprises adding a solution into the reservoir to wash away at least a portion of the gelatin composition.

### Claim 8 (depends on 1)

The method of claim 1, further comprising, after removing at least a portion of the gelatin composition, adding cell culture media into the reservoir comprising the plurality of cells.

### Claim 9 (depends on 1)

The method of claim 1, further comprising performing a high throughput assay with the plurality of cells in the reservoir.

### Claim 10 (depends on 1)

The method of claim 1, further comprising growing the plurality of cells in culture for one or more day(s).

### Claim 11 (depends on 1)

The method of claim 1, wherein the method provides a take rate of at least 50%.

### Claim 12 (depends on 1)

The method of claim 1, wherein the plurality of cells comprise at least 75% live cells based on the average number of cells in the reservoir at 1 week of culture.

### Claim 13

A method of printing a plurality of organoids, the method comprising:
depositing a gelatin composition into a plurality of reservoirs, wherein the gelatin composition is a solution and gelatin is dissolved in the gelatin composition;
adding a composition comprising a plurality of cells into the gelatin composition in each reservoir of the plurality of reservoirs, wherein the composition is added into the gelatin composition at a position below the surface of the gelatin composition and at least a portion of the composition and/or plurality of cells are suspended in the gelatin composition;
curing at least a portion of the composition comprising the plurality of cells in the gelatin composition to form an organoid in each reservoir; and
removing at least a portion of the gelatin composition in the plurality of reservoirs, thereby providing the plurality of organoids,
wherein the gelatin composition comprises gelatin at a concentration of about 1 mg/mL to about 15 mg/mL and, at 4° C., the gelatin composition has an elastic modulus of about 5 Pa to about 15 Pa at about 1% to about 100% strain, and
wherein the composition comprising the plurality of cells, at 4° C., has an elastic modulus of about 3 Pa to about 4 Pa at 1%-10% strain and the elastic modulus of the composition comprising the plurality of cells, at 4° C., is within an order of magnitude of the elastic modulus of the gelatin composition.

### Claim 14 (depends on 13)

The method of claim 13, wherein less than about 20% of the reservoirs are connected after adding the composition comprising the plurality of cells into the gelatin composition in each reservoir of the plurality of reservoirs.

### Claim 15 (depends on 13)

The method of claim 13, wherein the plurality of organoids, on average, have a uniform diameter in at least one dimension.

### Claim 16 (depends on 13)

The method of claim 13, wherein the plurality of organoids comprise at least 75% live cells based on the average number of cells in the plurality of organoids at 1 week of culture.

### Claim 17 (depends on 1)

The method of claim 1, wherein the composition suspended in the gelatin composition is in a substantially spherical form.

### Claim 18 (depends on 1)

The method of claim 1, wherein the gelatin in the gelatin composition is in a form consisting of dissolved gelatin.

### Claim 19 (depends on 13)

The method of claim 13, wherein the gelatin in the gelatin composition is in a form consisting of dissolved gelatin.