# US 12,600,951 B2 · Systems and methods for continuous cultivation and recovery of comestible non-human animal cells

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- Number: US 12,600,951 B2
- Document ID: US12600951B2
- Status: Granted
- Kind: B2
- Application number: 18/742,810
- Filed: 2024-06-13
- Published: 2025-12-18
- Granted: 2026-04-14
- Earliest claimed: 2024-06-13
- Assignees: Upside Foods, Inc.
- Applicants: Upside Foods, Inc.
- Inventors: Charles Knight Benton; Kesav Timiri Venkat Reddy
- Web page: https://patentgrep.com/patent/US12600951B2

## CPC

- C12N 5/0652: {Cells of skeletal and connective tissues; Mesenchyme}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to systems, apparatuses, and methods for continuous cultivation and recovery of comestible non-human animal cells in a suspension bioreactor. For example, in one or more implementations, the disclosed methods include cultivating non-human animal cells in suspension culture within a bioreactor vessel, removing a measure of suspension culture from the bioreactor vessel while continuing to provide cell culture media thereto, and recovering a first portion of cultivated non-human animal cells from the removed measure of suspension culture while returning a second portion of cultivated non-human animal cells to the bioreactor vessel. Also, in some implementations, the disclosed systems include at least one sterile output module configured to prevent backflow contamination when recovering cultivated cells and removing spent media during continuous cultivation of non-human animal cells according to the disclosed methods.

## Claims

### Claim 1

A method for producing comestible non-human animal cells, the method comprising:
cultivating suspension culture comprising non-human animal cells in a bioreactor vessel;
removing a measure of the suspension culture from the bioreactor vessel while providing cell culture media to the bioreactor vessel;
separating the removed measure of the suspension culture into a concentrated cell culture and a culture broth with cells removed;
recovering a first portion of the concentrated cell culture for harvest concurrent with returning a second portion of the concentrated cell culture to the bioreactor vessel without returning the first portion to the bioreactor vessel; and
processing cultivated non-human animal cells from the first portion of the concentrated cell culture to produce a cell-based food product.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
initially cultivating the suspension culture by providing the cell culture media to the bioreactor vessel for a first period of time; and
further cultivating the suspension culture within the bioreactor vessel by providing the cell culture media to the bioreactor vessel for a second period of time while:
removing the measure of the suspension culture from the bioreactor vessel without removing the measure of the suspension culture from a sterile environment comprising the bioreactor vessel and additional system components;
returning the second portion of the concentrated cell culture to the bioreactor vessel without removing the second portion from the sterile environment; and
recovering the first portion of the concentrated cell culture from the sterile environment.

### Claim 3 (depends on 2)

The method of claim 2, further comprising:
providing the cell culture media to the bioreactor vessel during the first period of time at a progressively increasing rate until a predetermined steady state rate; and
providing the cell culture media to the bioreactor vessel at the predetermined steady state rate during the second period of time.

### Claim 4 (depends on 2)

The method of claim 2, further comprising removing the measure of suspension culture from the bioreactor vessel at a continuous rate during the second period of time.

### Claim 5 (depends on 4)

The method of claim 4, further comprising recovering the first portion of the concentrated cell culture from the removed measure of the suspension culture at a progressively decreasing recovery rate while returning the second portion of the concentrated cell culture to the bioreactor vessel at a respectively increasing retention rate.

### Claim 6 (depends on 2)

The method of claim 2, further comprising:
separating the removed measure of the suspension culture into the concentrated cell culture and the culture broth with cells removed without removing the measure of suspension culture from the sterile environment.

### Claim 7 (depends on 1)

The method of claim 1, further comprising removing spent cell culture media from the first portion of the concentrated cell culture to recover the cultivated non-human animal cells.

### Claim 8 (depends on 1)

The method of claim 1, further comprising:
processing the culture broth with cells removed for reintroduction into the bioreactor vessel to further cultivate the non-human animal cells therein; and
returning the processed culture broth to the bioreactor vessel.

### Claim 9

A method for producing comestible non-human animal cells, the method comprising:
growing non-human animal cells in a suspension culture within a bioreactor for a first period of time; and
cultivating the suspension culture within the bioreactor for a second period of time by:
continuously providing cell culture media to the bioreactor for the second period of time;
drawing a measure of the suspension culture from the bioreactor during the second period of time;
separating the drawn measure of the suspension culture into a concentrated cell culture and a culture broth with cells removed;
returning a first portion of the concentrated cell culture to the bioreactor while continuously providing the cell culture media; and
recovering, during the second period of time, a second portion of the concentrated cell culture for harvest without returning the second portion to the bioreactor; and
processing cultivated non-human animal cells of the second portion of the concentrated cell culture to produce a cell-based food product.

### Claim 10 (depends on 9)

The method of claim 9, wherein growing the non-human animal cells within the bioreactor for the first period of time comprises providing cell culture media to the bioreactor, drawing and separating cell suspension culture from the bioreactor, returning concentrated cell culture to the bioreactor, and removing culture broth with cells removed until the non-human animal cells reach a target cell density within the suspension culture.

### Claim 11 (depends on 9)

The method of claim 9, further comprising:
returning at least a portion of the culture broth with cells removed to the bioreactor during the second period of time.

### Claim 12 (depends on 9)

The method of claim 9, further comprising maintaining a threshold cell density of the non-human animal cells within the bioreactor during the second period of time by continuously returning the first portion of the concentrated cell culture to the bioreactor while recovering the second portion of the concentrated cell culture.

### Claim 13 (depends on 12)

The method of claim 12, further comprising progressively increasing a retention rate of returning the first portion of the concentrated cell culture to the bioreactor during the second period of time.

### Claim 14 (depends on 9)

The method of claim 9, further comprising:
processing the culture broth with cells removed for reintroduction into the bioreactor to further cultivate the non-human animal cells therein; and
returning the processed culture broth to the bioreactor.

### Claim 15 (depends on 9)

The method of claim 9, further comprising:
recovering, utilizing a cell concentration device, cultivated non-human animal cells from the second portion of the concentrated cell culture; and
removing, utilizing the cell concentration device, spent cell culture media from the second portion of the concentrated cell culture.

### Claim 16 (depends on 15)

The method of claim 15, further comprising diluting the second portion of the concentrated cell culture prior to removing the spent cell culture media.

### Claim 17 (depends on 15)

The method of claim 15, further comprising preventing backflow contamination between the cell concentration device and lines back to the bioreactor.

### Claim 18 (depends on 17)

The method of claim 17, wherein preventing backflow contamination between the cell concentration device and the lines back to the bioreactor comprises alternating use and sterilization of at least two parallel output lines of a sterile output module.

### Claim 19 (depends on 11)

The method of claim 11, further comprising removing at least a second portion of the culture broth with cells removed as spent media.

### Claim 20 (depends on 9)

The method of claim 9, further comprising utilizing a bypass line to enable decoupling of respective line distances, flow rates, and residence times between a first zone circulating the suspension culture at a higher flow rate and a second zone drawing the measure of the suspension culture at a lower flow rate.