# US 11,299,700 B1 · Bioreactor containers and methods of growing hairy roots using the same

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- Number: US 11,299,700 B1
- Document ID: US11299700B1
- Status: Granted
- Kind: B1
- Application number: 17/180,489
- Filed: 2021-02-19
- Granted: 2022-04-12
- Earliest claimed: 2021-02-19
- Assignees: Acequia Biotechnology, LLC
- Applicants: Acequia Biotechnology, LLC
- Inventors: Rocio S. Honigmann; Jaime Flores-Riveros; Martin Sierra-Honigmann
- Web page: https://patentgrep.com/patent/US11299700B1

## CPC

- A01G 31/06: Hydroponic culture on racks or in stacked containers
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12M 23/04: {Flat or tray type, drawers (C12M23/10, C12M23/12, C12M23/16 take precedence)}
- C12M 23/06: {Tubular (C12M23/08, C12M23/16 take precedence)}
- C12M 23/08: {Flask, bottle or test tube}
- C12M 25/06: {Plates; Walls; Drawers; Multilayer plates}
- C12M 27/14: {Rotation or movement of the cells support, e.g. rotated hollow fibers}
- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- Y02P 60/21: Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
- A01G: HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING (picking of fruits, vegetables, hops or the like A01D46/00; propagating unicellular algae C12N1/12)
- 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)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Bioreactors and methods of stimulating continuous growth of hairy root biomass in such bioreactors are provided.

## Claims

### Claim 1

A bioreactor container, comprising:
a container housing having a plurality of side portions, a bottom portion, and a top portion, wherein the top portion is sealably connectable to the plurality of side portions to define an internal chamber;
an internal wall in at least one of the plurality of side portions that defines a media exchange aperture; and
a mesh scaffolding disposable within the internal chamber of the container housing, wherein the mesh scaffolding further comprises:
a series of alternating incline sections and decline sections each connected by a vertex, which form a plurality of channels spaced across the mesh scaffolding;
wherein a relative position of the incline sections and decline sections are maintained by a support member that runs through opposing longitudinal edges of the mesh scaffolding.

### Claim 2 (depends on 1)

The bioreactor container of claim 1, further comprising:
a first tubing member sealably connectable to the media exchange aperture and extending inside the container housing; and
a second tubing member sealably connectable to the media exchange aperture and extending outside the container housing.

### Claim 3 (depends on 1)

The bioreactor container of claim 1, wherein the mesh scaffolding further comprising support members, which further include limiting members to contain movement of the mesh scaffolding along the support members.

### Claim 4 (depends on 1)

The bioreactor container of claim 1, wherein the media exchange aperture is configured to dispense and suction culture media.

### Claim 5 (depends on 2)

The bioreactor container of claim 2, wherein the first tubing member comprises an end portion that runs parallel to the internal wall.

### Claim 6 (depends on 2)

The bioreactor container of claim 2, wherein the first tubing member comprises an end portion juxtaposed to a corner at the bottom portion of the container housing.

### Claim 7 (depends on 1)

The bioreactor container of claim 1, wherein the media exchange aperture is only present on one of the plurality of side portions.

### Claim 8 (depends on 1)

The bioreactor container of claim 1, wherein the bioreactor has a single media exchange aperture.

### Claim 9 (depends on 1)

The bioreactor container of claim 1, wherein the top portion, when sealably connected to the plurality of side portions, is parallel to the bottom portion.

### Claim 10 (depends on 1)

The bioreactor container of claim 1, wherein the mesh scaffolding is folded in an accordion-like fashion.

### Claim 11 (depends on 1)

A method of growing hairy root tissue, comprising:
placing a plurality of hairy root tissue samples on a bottom portion of the bioreactor container of claim 1;
positioning a mesh scaffolding on top of the hairy root tissue samples so as to cover the samples, wherein the mesh scaffolding includes a series of alternating incline sections and decline sections each connected by a vertex, which form a plurality of channels spaced across the mesh scaffolding;
sealing the internal chamber of the bioreactor container by releasably connecting a removable top portion to the plurality of side portions;
adding a culture medium to the internal chamber of the bioreactor container through a tube sealably connected to a media exchange aperture formed by an internal wall in one of the plurality of side portions of the bioreactor container; and
culturing the hairy root tissue samples.

### Claim 12 (depends on 11)

The method of claim 11, further comprising moving the bioreactor container according to a motion profile.

### Claim 13 (depends on 12)

The method of claim 12, further comprising adding an elicitor to the bioreactor container.

### Claim 14 (depends on 13)

The method of claim 13, further comprising removing the hairy root tissue from the bioreactor container.

### Claim 15 (depends on 11)

The method of claim 11, wherein the hairy root tissue samples are cultured for 3-12 weeks.

### Claim 16 (depends on 1)

A system of bioreactor containers, comprising at least two bioreactor containers according to claim 1.

### Claim 17 (depends on 16)

The system of bioreactor containers of claim 16, wherein each bioreactor container further comprises:
a first tubing member sealably connectable to the media exchange aperture and extending inside the container housing; and
a second tubing member sealably connectable to the media exchange aperture and extending outside the container housing.

### Claim 18 (depends on 1)

A method of growing hairy root tissue, comprising:
placing a plurality of hairy root tissue samples on a bottom portion of the bioreactor container of claim 1;
positioning the mesh scaffolding on top of the hairy root tissue samples so as to cover the samples;
sealing the internal chamber of the bioreactor container by releasably connecting a removable top portion to the plurality of side portions;
adding a culture medium to the bioreactor container through a tube sealably connected to a media exchange aperture formed by an internal wall in one of the plurality of side portions of the bioreactor container;
loading the bioreactor container onto an apparatus for stimulating plant cultures;
moving the bioreactor containers on the apparatus according to a motion profile;
and culturing the hairy root tissue samples.

### Claim 19 (depends on 18)

The method of claim 18, further comprising adding an elicitor to the bioreactor container.

### Claim 20 (depends on 19)

The method of claim 19, further comprising removing the hairy root tissue from the bioreactor container.

### Claim 21 (depends on 18)

The method of claim 18, wherein the hairy root tissue samples are cultured for 3-12 weeks.

### Claim 22 (depends on 18)

The method of claim 18, wherein the apparatus for stimulating plant cultures, comprises
a frame including a support member;
one or more trays movably connected to the support member;
wherein each tray comprises:
a bottom portion having a first edge and a second edge;
a first flange member extending from the first edge above the bottom portion; and
a second flange member extending from the second edge above the bottom portion;
wherein each tray is configured to move between a first angled position, where the first edge is below the second edge, and a second angled position, wherein the second edge is below the first edge; and
a motion mechanism connected to the one or more trays and configured to move the one or more trays between the first angled position and the second angled position according to a motion profile configured to stimulate growth of the plant cultures.