US-12620244-B2 - Methods and devices for live cell imaging analysis

US12620244B2US 12620244 B2US12620244 B2US 12620244B2US-12620244-B2

Abstract

Provided herein are methods for analysis of target cells on a population or individual basis, including before and after contact with a stimulus in order to determine the effect of such stimulus on the target cells. Also provided are devices for performing such methods. The analysis methods involve identifying and measuring or tracking morphological changes that occur in target cells over a period of time. Tracking is accomplished using imaging systems capable of imaging target cells individually over a period of time either continuously or at discrete intervals of time.

Inventors

Lee L. Rubin
Yasujiro Kiyota
Chieko NAKADA
Keiichi Niikura
Kathleen L. Pfaff

Assignees

PRESIDENT AND FELLOWS OF HARVARD COLLEGE
NIKON CORPORATION

Dates

Publication Date: 20260505
Application Date: 20200720

Claims (12)

1 . A method for manipulating cells in an incubator having an imaging device, the method comprising: executing a control program using processor circuitry, wherein the control program corresponds to executable instructions encoded in a storage medium that, when executed by the processor circuitry, cause the processor circuitry to: control the incubator to culture a neuronal cell population in vitro, wherein a neuron of the neuronal cell population has a cell body and a number of nodes, and wherein a node is a junction between the cell body and a neurite that extends from the cell body; control, while the incubator is culturing the neuronal cell population in vitro, the imaging device of the incubator to repeatedly acquire an image of a neuron in the neuronal cell population during cell differentiation, the neuron having a cell body and a number of nodes, wherein a node is a junction between the cell body and a neurite extending from the cell body; detect the number of nodes of the neuron by analyzing morphological features based on the image of the neuron; compare the number of nodes of the neuron to a predetermined number to analyze a state of the neuron to obtain a comparison result; monitor the neuron in the neuronal cell population for a determination that the neuron is in a healthy state; determine that the neuron is in the healthy state if the comparison result indicates that the number of nodes exceeds the predetermined number, and determine that the neuron is in an unhealthy state if the comparison result indicates the number of nodes is less than the predetermined number; and upon determining that the neuron is in the healthy state, contacting the neuron with a stimulus.
2 . The method of claim 1 , wherein executing the control program to cause the processor circuitry to detect a number of nodes of the neuron based on analysis of the image further comprises analyzing a neuronal cell population by comparing the number of nodes of each neuron of the neuronal cell population with the predetermined number.
3 . The method of claim 1 , wherein the neuron is a first neuron in the neuronal cell population, and wherein executing the control program to cause the processor to culture the neuronal cell population further comprises: executing the control program to cause the incubator to culture the neuronal cell population until a percentage of the neuronal cell population has a defined phenotype.
4 . The method of claim 1 , further comprising: executing the control program to cause the processor circuitry to: receive information about a stimulus from an input device; and contact the neuron with the stimulus based on the determined state of the neuron, wherein the stimulus is a chemical, electrical, electromagnetic, mechanical, or other agent that can be administered to the neuron.
5 . The method of claim 4 , further comprising executing the control program to cause the processor circuitry to determine changes in the number of nodes of the neuron by measuring and comparing the number of nodes of the neuron pre-and post-exposure to the stimulus.
6 . The method of claim 5 , further comprising executing the control program to cause the processor circuitry to measure and compare the number of nodes of the neuron post-exposure to the stimulus in both neurons in a healthy state and neurons in an unhealthy state.
7 . The method of claim 4 , wherein the neuron is obtained by in vitro differentiation of a pluripotent stem cell.
8 . The method of claim 7 , wherein the pluripotent stem cell is an induced pluripotent stem cell derived from a human subject having a neurological disorder.
9 . The method of claim 4 , further comprising: executing the control program to cause the processor circuitry to measure a time between when the neuron is contacted with the stimulus and when changes in the number of nodes of the neuron is detected.
10 . The method of claim 1 , further comprising monitoring additional neurons in the neuronal cell population to determine whether more neurons than a threshold portion of the neuronal cell population are in the healthy state.
11 . The method of claim 1 , further comprising executing the control program to cause the processor circuitry to: calculate a period of time in which the neuron is in a healthy condition.
12 . The method of claim 1 , further comprising executing the control program to cause the processor circuitry to: control the imaging device to repeatedly acquire an image of the neuron in the neuronal cell population after cell differentiation using the imaging device over a period of time during the in vitro culturing by the incubator; detect the number of nodes of the neuron by analyzing morphological features based on the image of the neuron; compare the number of nodes of the neuron to a predetermined number to analyze a state of the neuron to obtain a comparison result; and determine that the neuron is in a healthy state if the comparison result indicates that the number of nodes exceeds the predetermined number, and determine that the neuron is in an unhealthy state if the comparison result indicates the number of nodes is less than the predetermined number.

Description

RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 120 as a continuation of U.S. application Ser. No. 15/565,611 filed Oct. 10, 2017, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2016/026833, filed Apr. 8, 2016, which was published under PCT Article 21(2) in English, and which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/145,730 filed Apr. 10, 2015, and U.S. Provisional Application No. 62/183,703 filed Jun. 23, 2015, both entitled “METHODS AND DEVICES FOR LIVE CELL IMAGING ANALYSIS.” Each of the foregoing applications are incorporated by reference herein in the entirety. BACKGROUND OF INVENTION The current drug discovery system for diseases of the nervous system (and most other disorders) relies heavily on animal models. Standard in vitro assays trying to identify drugs to treat neurodegenerative diseases employ single measurements of cell survival in which live healthy and diseased cells are counted after a fixed time period. SUMMARY OF INVENTION The drug discovery assays of the prior art are considered sub-optimal for a number of reasons. Animal models may only partially mimic the human disease they are intended to model. In addition, these in vivo models are inefficient both in terms of cost and time. In vitro assays typically only assess a single characteristic and then only once at or near the end of simulated disease progression. A classic example of this is assessment of cell viability (or alternatively cell death) following exposure to a drug or other stimulus. Such measurements fail to take into consideration earlier events that may be involved in or may be manifestations of disease onset and/or progression. They also do not take into account cell heterogeneity that exists in cell culture, including particularly in stem cell derived cultures. The invention addresses various shortcomings of the prior art methods. The invention provides inter alia methods and devices for analyzing the effect(s) of one or more stimuli on a population of cells in vitro by identifying, assessing, and/or monitoring one or more changes in the population of cells over a period of time. Such changes may be morphological changes in target cells, including changes that can be detected, measured and monitored over time using an imaging system such as a microscope system. The invention further provides methods for evaluating status of a population of cells, according to one or more markers, and determining based on those markers the optimal time to contact the population of cells with a stimulus. Such methods are therefore individualized for a given population of cells (e.g., a cell line) in that stimulus is added only when a certain, typically pre-established, threshold is met. As discussed in greater detail below, the threshold may be a healthy or an unhealthy threshold. The methods differ from the prior art methods in a number of ways. First, they measure an effect other than cell survival or cell death. The measured effect may be related to cell viability or it may not be related to cell viability. The latter situation is particularly helpful when the disease being studied is characterized by a dysfunction in a target cell population rather than by death of that target population. Second, the methods provided herein measure such effect over a length of time, whether continuously or at discrete time intervals, rather than at only a single time point at or near the end of the assay time-course. In this way, the methods provide a higher degree of resolution and understanding of earlier disease markers and the impact that various stimuli (including drug candidates) have on such markers. The ability to track earlier markers also potentially results in shorter assays, thereby increasing the efficiency of such assays both in terms of time and cost. Finally, tracking of earlier effects and the ability of a stimulus to impact an earlier event can lead to identification of stimuli (and potentially therapeutics) that can be used at early stages of disease progression, when impact may be greatest. The invention therefore provides, in a general sense, screening methods (or screening assays) for determining the effect of a stimulus on a target cell population. These methods comprise the steps of obtaining a baseline measurement of a marker (such as a morphological marker) of a cell population, exposing the cell population to a stimulus, measuring the marker continuously or at discrete time intervals in order to identify and/or quantitate changes in the marker over time, wherein a change to the marker relative to baseline indicates the stimulus altered the development of the cell population. In some embodiments, the cell population is a diseased cell population. Such populations may be patient-specific cell lines. In some instances, such cell lines may be generated through directed, lineage-specific, differentiation of patient-sp