# US 2023/0056414 A1 · 3d cell culture gel kit and 3d cell culture method using the same

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- Number: US 2023/0056414 A1
- Document ID: US20230056414A1
- Status: Published application
- Kind: A1
- Application number: 17/759,124
- Filed: 2020-08-26
- Published: 2023-02-23
- Earliest claimed: 2020-08-26
- Assignees: Gecoll Biomedical Co., Ltd.
- Applicants: Gecoll Biomedical Co., Ltd.
- Inventors: YU-CHUN WU; CHING-WEN LIU; WEI-YU LIN
- Web page: https://patentgrep.com/patent/US20230056414A1

## CPC

- C12N 5/0062: {General methods for three-dimensional culture}
- C08J 3/24: Crosslinking, e.g. vulcanising, of macromolecules (mechanical aspects B29C35/00; crosslinking agents C08K)
- C08L 5/00: Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- C12N 2513/00: 3D culture
- C12N 2533/54: Collagen; Gelatin
- 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)
- C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- 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)

## Abstract

A 3D cell culture gel kit and a 3D cell culture method using the same are provided. The 3D cell culture gel kit includes a gel material A, a buffer solution C, and a buffer solution D. The 3D cell culture method includes the steps of adding cells into a mixed solution containing the gel material A and setting the mixed solution at low temperature to get gel containing the cells. Then adding the buffer solution C to the gel for performing crosslinking. Next removing the buffer solution C and adding a growth medium. Let stand until the cells form spheroids in the gel. Moreover, the buffer solution D is used to dissolve the gel and the cells cultured are taken out for analysis. Thereby the 3D cell culture gel kit is convenient to use and suitable for 3D culture of a plurality of cell lines.

## Claims

### Claim 1

A three-dimensional (3D) cell culture gel kit comprising:
a gel material A which contains 0.5-3 wt % sodium alginate, 2.5-15 wt % gelatin, 0.5-2 wt % 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and the remaining percentage of pure water;
a buffer solution C which contains 0.2-10 wt % salts of divalent cations, 0.01-1 wt % the HEPES buffer solution, and the remaining percentage of pure water; and
a buffer solution D having 1-10 wt % disodium ethylenediaminetetraacetate, 0.1-1 wt % sodium hydroxide (NaOH), 0.05-1 wt % the HEPES buffer solution, and the remaining percentage of pure water.

### Claim 2 (depends on 1)

The 3D cell culture gel kit as claimed in claim 1, wherein the salts of is the divalent cation of the buffer solution C is selected from the group consisting of strontium chloride, calcium phosphate, calcium chloride, calcium sulfate, and combinations thereof.

### Claim 3 (depends on 1)

The 3D cell culture gel kit as claimed in claim 1, wherein the HEPES buffer solution is 0.5-3 M HEPES buffer solution.

### Claim 4 (depends on 1)

The 3D cell culture gel kit as claimed in claim 1, wherein the 3D cell culture gel kit further includes a thermal conductive sheet.

### Claim 5 (depends on 1)

A 3D cell culture method using the 3D cell culture gel kit as claimed in claim 1 comprising the steps of:
(a) suspending cells in a growth medium to get a cell suspension and then mixing the cell suspension with the gel material A in a volume ratio of 1:1 to obtain a mixed solution;
(b) arranging a Petri dish at a cryogenic component, adding the mixed solution into wells of the Petri dish, and allowing the mixed solution to stand for 1-7 minutes to form gel;
(c) adding buffer solution C to the gel and standing for 10-20 minutes to carry out crosslinking;
(d) replacing the buffer solution C with the growth medium; and
(e) arranging the Petri dish at a temperature required until the cells form spheroids in the gel while the growth medium in the Petri dish needs to be replaced once a day.

### Claim 6 (depends on 5)

The method as claimed in claim 5, wherein the gel material A contains 0.5-2 wt % sodium alginate, 2.5-15 wt % gelatin, 0.5-2 wt % 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and the remaining percentage of pure water; wherein the buffer solution C contains 0.2-10 wt % salts of divalent cations, 0.01-1 wt % HEPES buffer solution, and the remaining percentage of pure water.

### Claim 7 (depends on 5)

The method as claimed in claim 5, wherein the method further includes a step (f) run after the step (e); the step (f) is washing the spheroids with phosphate buffer solution, removing the phosphate buffer solution, and then adding buffer solution D and mixing the buffer solution D with the spheroids evenly; next reacting at room temperature for 5 minutes to make the gel dissolve; wherein the buffer solution D contains 1-10 wt % disodium ethylenediaminetetraacetate, 0.1-1 wt % sodium hydroxide, 0.05-1 wt % the HEPES buffer solution, and the remaining percentage of pure water.

### Claim 8 (depends on 7)

The method as claimed in claim 7, wherein the method further includes a step (g) run after the step (f); the step (g) is washing dissolved gel solution with the phosphate buffer solution and collecting cell precipitate for analysis of cells in the cell precipitate after centrifugation and removal of supernatant.

### Claim 9 (depends on 5)

The method as claimed in claim 5, wherein the cryogenic component in the step (b) is obtained by a thermal conductive sheet placed and cooled on a cryogenic device; wherein temperature required is 30-37 ° C.; wherein cell density of the mixed solution is 104-107 cells/mL.

### Claim 10 (depends on 6)

The method as claimed in claim 6, wherein the salts of the divalent cation of the buffer solution C is selected from the group consisting of strontium chloride, calcium phosphate, calcium chloride, calcium sulfate, and combinations thereof.