# US 12,595,458 B2 · Device and method for determining a viability and/or a cell count of biological cells in a suspension by means of collimated transmission

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- Number: US 12,595,458 B2
- Document ID: US12595458B2
- Status: Granted
- Kind: B2
- Application number: 17/925,897
- Filed: 2021-05-03
- Published: 2023-06-15
- Granted: 2026-04-07
- Earliest claimed: 2020-05-20
- Assignees: Sartorius Stedim Biotech GmbH; Stitftung für Lasertechnologien in der Medizin und Me technik an der Universität Ulm
- Applicants: Sartorius Stedim Biotech GmbH; Stiftung für Lasertechnologien in der Medizin und Meßtechnik ań der Universität Ulm (Stiftung Bürgerliches Recht)
- Inventors: Steffen Nothelfer; Alwin Kienle; Florian Foschum; Marek Hoehse
- Web page: https://patentgrep.com/patent/US12595458B2

## CPC

- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)}
- 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

Devices and methods are provided for determining viability and/or a cell counts of biological cells in a cell suspension culture using collimated transmission. Devices can include an illumination source for generating an electromagnetic illumination beam; beam manipulation means for collimating the illumination beam; and a detection unit for detecting an electromagnetic transmission beam being a portion of the collimated illumination beam which has been transmitted through a sample of the cell suspension culture.

## Claims

### Claim 1

A device for determining a viability and/or a cell count of biological cells in a cell suspension culture by means of collimated transmission, comprising:
an illumination source for generating an electromagnetic illumination beam;
beam manipulation means for collimating the illumination beam; and
a detection unit for detecting an electromagnetic transmission beam being a portion of the collimated illumination beam which has been transmitted through a sample of the cell suspension culture,
wherein the beam manipulation means are configured to manipulate the illumination beam and/or the transmission beam such that an effective acceptance angle θa of the collimated transmission is equal to or below a specified threshold value, wherein the effective acceptance angle θa is defined as the sum of:
a maximal illumination beam divergence angle θ1, of the collimated electromagnetic illumination beam, and
a maximal transmission beam detection angle θ2 of the transmission beam.

### Claim 2 (depends on 1)

The device according to claim 1, wherein:
the beam manipulation means are configured to collimate the illumination beam such that the illumination beam has a divergence angle of less than 1.5 degrees; and/or
the beam manipulation means are configured to manipulate the transmission beam such that the transmission beam has a divergence angle of less than 1.5 degrees, and/or
the beam manipulation means are configured to manipulate the illumination beam and/or the transmission beam such that an effective acceptance angle θa of the collimated transmission is equal to or less than 3 degrees.

### Claim 3 (depends on 1)

The device according claim 1, wherein the beam manipulation means comprise at least one of the following:
an illumination fiber for guiding the illumination beam;
an illumination beam collimation unit for collimating the illumination beam;
a transmission beam focusing unit for focusing the transmission beam; and
a detection fiber for guiding the transmission beam to the detection unit.

### Claim 4 (depends on 3)

The device according to claim 3, wherein the illumination fiber and/or the detection fiber have a numerical aperture smaller than 0.02.

### Claim 5 (depends on 3)

The device according to claim 3, wherein the illumination beam collimation unit and/or the transmission beam focusing unit comprises at least one of the following:
an aperture, a refractive focusing unit, and a reflective focusing unit.

### Claim 6 (depends on 1)

The device according to claim 1, wherein:
the beam manipulation means comprise an illumination beam collimation unit, the illumination beam collimation unit comprising an illumination fiber and an illumination focusing optics,
wherein the illumination fiber is configured to guide the illumination beam to the illumination focusing optics,
wherein the illumination focusing optics is arranged between an end of the illumination fiber and the sample such that a distance between the end of the illumination fiber and the illumination focusing optics is equal to a focal length (f1) of the illumination focusing optics; and/or
the beam manipulation means comprise a transmission beam focusing unit, the transmission beam focusing unit comprising a detection focusing optics and a detection fiber,
wherein the detection fiber is configured to guide the transmission beam to the detection unit,
wherein the detection focusing optics is arranged between the sample and an end of the detection fiber such that a distance between the detection focusing optics and the end of the detection fiber is equal to a focal length (f2) of the detection focusing optics.

### Claim 7 (depends on 6)

The device according to claim 6, wherein the effective acceptance angle θa is given by the following equation:
θ
a
=
tan
-
1
(
d
1
f
1
)
+
tan
-
1
(
d
2
f
2
)
.
where d1 denotes an inner diameter of the illumination fiber (2), d2 an inner diameter of the detection fiber, f1 the focal length of the illumination focusing optics, and f2 the focal length of the detection focusing optics.

### Claim 8 (depends on 1)

The device according to claim 1, wherein:
the illumination source is a polychromatic light source and the detection unit is a spectrometer; or
the frequency of the illumination source is tuneable and the detection unit is a broadband detector or a tunable monochromatic detector.

### Claim 9 (depends on 1)

The device according to claim 1, further comprising:
an evaluation unit for determining the viability, a viable cell count and/or a total cell count of the biological cells based on the detected transmission beam,
wherein in particular, the evaluation unit is configured to determine an extinction spectrum based on the detected transmission beam and to determine the viability, the viable cell count and/or the total cell count based on the extinction spectrum by comparing the determined extinction spectrum with reference extinction spectra.

### Claim 10 (depends on 9)

The device according to claim 9, wherein the reference extinction spectra are modelled based on first principles by solving Maxwell's equations and/or using Mie's theory.

### Claim 11 (depends on 9)

The device according to claim 9, wherein the reference extinction spectra are obtained by measurements carried out on samples with known properties.

### Claim 12 (depends on 1)

The device according to claim 1, comprising a sample container for containing a sample of the cell suspension culture.

### Claim 13 (depends on 1)

A bioreactor comprising the device according to claim 1.

### Claim 14

A method for determining a viability and/or a cell count of biological cells in a cell suspension culture by means of a collimated transmission, the method comprising:
illuminating a sample of the cell suspension culture with a collimated electromagnetic illumination beam;
detecting an electromagnetic transmission beam being a portion of the collimated electromagnetic illumination beam which has been transmitted through the sample of the cell suspension culture; and
determining the viability and/or the cell count of the biological cells based on the detected transmission beam,
wherein the illumination beam and/or the transmission beam are/is manipulated such that an effective acceptance angle θa of the collimated transmission is equal to or below a specified threshold value, and
wherein the effective acceptance angle θa is defined as the sum of:
a maximal illumination beam divergence angle θ1 of the collimated electromagnetic illumination beam, and
a maximal transmission beam detection angle θ2 of the transmission beam.

### Claim 15 (depends on 1, 14)

The method of claim 14, wherein the collimated transmission is performed by a device according to claim 1.