# US 12,215,344 B2 · Engineered multicellular ciliated organisms and kinematic self-replication thereof

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- Number: US 12,215,344 B2
- Document ID: US12215344B2
- Status: Granted
- Kind: B2
- Application number: 17/647,847
- Filed: 2022-01-12
- Published: 2022-07-14
- Granted: 2025-02-04
- Earliest claimed: 2021-01-12
- Assignees: Trustees of Tufts College; University of Vermont
- Applicants: Trustees of Tufts College; University of Vermont
- Inventors: Michael Levin; Douglas J. Blackiston; Kelly A. McLaughlin; Josh Bongard; Sam Kriegman
- Web page: https://patentgrep.com/patent/US12215344B2

## CPC

- C12N 5/06: {Animal cells or tissues; Human cells or tissues (preservation of excised living parts A01N1/10)}
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 35/08: {Chemical, biochemical or biological means, e.g. plasma jet, co-culture}
- 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}

## Abstract

Disclosed are engineered multicellular organisms. The disclosed organisms comprise an aggregate of ciliated cells, and the organisms move when the ciliated cells are actuated. The engineered multicellular organisms also are capable of kinematic self-replication. The engineered multicellular organisms are capable of moving a plurality of dissociated ciliated cells into piles of ciliated cells which form a multicellular organism comprising an aggregate of ciliated cells which moves when the ciliated cells are actuated. Also disclosed are systems and methods for designing, preparing, and utilizing the engineered multicellular organisms.

## Claims

### Claim 1

An engineered multicellular organism comprising an aggregate of ciliated Xenopus laevis (X. laevis) cells derived from fertilized X. laevis embryonic cells, wherein the cilia of the ciliated cells move the organism at a rate of at least 15 microns/second in an aqueous environment.

### Claim 2 (depends on 1)

The organism of claim 1, wherein the organism consists of biological material and/or does not comprise any inorganic material.

### Claim 3 (depends on 1)

The organism of claim 1, wherein the organism comprises a sensor for detecting a target molecule.

### Claim 4 (depends on 1)

The organism of claim 1, wherein the cells of the organism self-assemble.

### Claim 5 (depends on 1)

The organism of claim 1, wherein the organism has an effective diameter of about 100-500 microns.

### Claim 6 (depends on 1)

The organism of claim 1, wherein the organism does not comprise neural cells or neural tissue.

### Claim 7 (depends on 1)

The organism of claim 1, wherein the ciliated cells are X. laevis epidermal cells.

### Claim 8 (depends on 1)

The organism of claim 1, wherein the ciliated cells are engineered to express a heterologous molecule.

### Claim 9 (depends on 8)

The organism of claim 8, wherein the heterologous molecule is a reporter molecule.

### Claim 10 (depends on 8)

The organism of claim 8, wherein the heterologous molecule is an enzyme that metabolizes a target substrate.

### Claim 11 (depends on 8)

The organism of claim 8, wherein the heterologous molecule is a receptor for a target ligand.

### Claim 12 (depends on 1)

The organism of claim 1, wherein the aggregate of cells reaggregates after the aggregate is subjected to deaggregation.

### Claim 13 (depends on 1)

The organism of claim 1, wherein the organism is capable of moving a target object.

### Claim 14 (depends on 1)

The organism of claim 1, wherein the organism comprises a cavity for capturing and/or transporting a target object.

### Claim 15 (depends on 1)

A plurality of the organism of claim 1, wherein the plurality exhibits collective and/or coordinated behavior.

### Claim 16 (depends on 15)

The plurality of claim 15, wherein the collective and/or coordinated behavior is collective and/or coordinated movement.

### Claim 17 (depends on 1)

A method for preparing the engineered multicellular organism of claim 1 or a plurality thereof, the method comprising explanting stem cells derived from fertilized X. laevis embryonic tissue and culturing the explanted cells under conditions in which the cultured, explanted cells form the engineered multicellular organism or the plurality thereof, wherein the conditions comprise culturing the explanted cells at 14° C.