# US 2020/0399589 A1 · Macrotumor engineering

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- Number: US 2020/0399589 A1
- Document ID: US20200399589A1
- Status: Published application
- Kind: A1
- Application number: 16/896,278
- Filed: 2020-06-09
- Published: 2020-12-24
- Earliest claimed: 2019-06-19
- Assignees: Purdue Research Foundation
- Applicants: Purdue Research Foundation
- Inventors: Sophie Andree Lelievre; Rahim Rahimi; Tim Kwok
- Web page: https://patentgrep.com/patent/US20200399589A1

## CPC

- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium
- C12N 2501/315: Prolactin
- C12N 2501/39: Steroid hormones
- C12N 2501/392: Sexual steroids
- C12N 2533/30: Synthetic polymers
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/78: Cellulose
- C12N 2533/80: Hyaluronan
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- 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}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A collagen-fibronectin-based method enables the production of tumors of centimeter size with recognizable pathological traits. The method supports reproducing tumor heterogeneity with preinvasive and invasive phenotypes and stacking of tumor portions using a paper-scaffold with 80 μm-punched holes for larger nodule creation and easy separation of tumor portions for analysis. Macrotumors are convenient for testing drug delivery and therapeutic tools that necessitate a minimum tumor size relevant to in vivo.

## Claims

### Claim 1

A multilayer stacking for culturing macrotumors, wherein the multilayer stacking comprises a plurality of cell culture amenable substrates, wherein each substrate has a plurality of pores with a diameter of 60-100 μm, wherein the density of the pores on each substrate is 2-8 pores/mm2, and wherein each substrate is pre-coated with a solution comprising one or more extracellular matrix.

### Claim 2 (depends on 1)

The multilayer stacking of claim 1, wherein the multilayer stacking comprises 2-4 cell culture amenable substrates.

### Claim 3 (depends on 1)

The multilayer stacking of claim 1, wherein said cell culture amenable substrate comprises polymeric material.

### Claim 4 (depends on 3)

The multilayer stacking of claim 3, wherein the polymeric material is cellulose acetate, polystyrene, polyurethane, polytetrafluoroethylene (PTFE), polyvinylchloride, polycarbonate, SU-8, or any combination thereof.

### Claim 5 (depends on 1)

The multilayer stacking of claim 1, wherein the multilayer stacking has a stiffness of 500-8,000 Pa if measured by indentation of unconstrained samples.

### Claim 6 (depends on 1)

The multilayer stacking of claim 1, wherein said one or more extracellular matrix is collagen I, fibronectin, collagen III, collagen IV, laminins, hyaluronic acid, heparan sulfate proteoglycan, or a combination thereof.

### Claim 7

A method of preparing a microtumor, comprising
a) providing a pellet comprising a plurality of microtumors of 50-800 microns;
b) providing a first cell culture amenable substrate, wherein the first substrate has a plurality of pores with a diameter of 60-100 μm, wherein the density of the pores on each substrate is 2-8 pores/mm2, and the first substrate is pre-coated with a solution comprising one or more extracellular matrix, and depositing the pellet comprising a plurality of microtumors onto the first cell culture amenable substrate;
c) adding an extracellular matrix solution onto the pellet deposited on the first substrate to allow the pellet to be embedded by the extracellular matrix solution;
d) providing a second cell culture amenable substrate, wherein the second substrate has a plurality of pores with a diameter of 60-100 μm, wherein the density of the pores on each substrate is 2-8 pores/mm2, and the second substrate is pre-coated with a solution comprising one or more extracellular matrix, and placing the second cell culture amenable substrate onto the extracellular matrix embedded microtumors deposited on the first cell culture amenable substrate;
e) adding a solution comprising one or more extracellular matrix solution onto the pellet of deposited on the second substrate to allow the pellet to be embedded by the extracellular matrix solution;
f) optionally repeating steps a)-e) to provide a total of 2-5 cell culture amenable substrates, wherein one pellet comprising a plurality of microtumors is deposited between two adjacent substrates;
g) incubating the obtained multilayer stacking in a cell culture medium.

### Claim 8 (depends on 7)

The method of claim 7, wherein microtumors can be same or different microtumors to provide homogeneous or heterogeneous macrotumors.

### Claim 9 (depends on 7)

The method of claim 7, wherein microtumors may be from cells representing different carcinomas.

### Claim 10 (depends on 7)

The method of claim 7, wherein said one or more extracellular matrix is collagen I, fibronectin, collagen III, collagen IV, laminins, hyaluronic acid, heparan sulfate proteoglycan, or a combination thereof.