CA2607217A1 - Method of cultivating a cell culture is an automated cell culture system as well as an automated cell culture system - Google Patents
Method of cultivating a cell culture is an automated cell culture system as well as an automated cell culture system Download PDFInfo
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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Abstract
The present invention relates to a method of cultivating a cell culture in an automated cell culture system, and to an automated cell culture system of this kind, data concerning the condition of cells in the cell culture being acquired, and at least one culturing condition being regulated in accordance with the acquired condition of the cells in the cell culture, and/or at least one operating step determined to be necessary in the cell cultivation process being initiated or carried out.
Description
Method of cultivating a cell culture in an automated cell culture system as well as an automated cell culture system The present invention relates to a method of cultivating a cell culture in an automated cell culture system and to an automated cell culture system. In particular, the present invention relates to a device and a method of regulating cell culturing conditions and cell cultivation operating steps subject to the condition of the biological cells.
Cell cultures are often used in pharmaceutical and biotechnological industries. In particular for ethical and economical reasons, animal experiments have been increasingly replaced by cell culture systems and cell culture technologies in recent years. For example, the European Union and the OECD also recommend the replacement of animal experiments by cell tests without animal experiments for testing medicine, chemicals and cosmetics. The employment of cell cultures in the search for new active principles and active agents in the pharmaceutical and pesticide sectors has therefore meanwhile become inevitable.
Moreover, a number of in-vitro test methods in the field of toxicology has been increasingly adopted in regulatory proceedings. Thus, in the pharmaceutical and pesticide sectors, too, the use of cell cultures is no longer dispensed with.
However, a significance of the data obtained with cell cultures is at present still limited by the existing analysis technique. Moreover, common manual methods in cell cultivation are time consuming and costly, and their results moreover highly depend on the user.
Due to this fact, initiatives have been started recently for defining minimum requirements on cell and tissue cultures and for thus ensuring the significance, comparability and reproducibility of in-vitro works. As a result, analogously to the Good Laboratory Practice (GLP) directives, principles for cell culture works have been passed with the designation Good Cell Culture Practice (GCCP).
These directives, however, have no influence on the fact that today, as a rule, cell cultures are still cultivated employing a lot of laboratory personnel. The cells are, as a rule, cultivated in Petri dishes, micro-titre plates or in cell culture bottles. In most cases, the steps necessary for cell cultivation, such as initial inoculation of the cells, change of the medium or passage of the cells, are performed merely manually. Moreover, the monitoring of the cells is discontinuous.
Changes of the pH value of the medium are observed by added color indicators.
The medium is changed at regular intervals, depending on the growth rate of the respective cells of the cell cultures (by manual control after removal from an incubator) to ensure sufficient supply of the cells with nutrients.
As one can draw conclusions from the results of cell cultivation, i.e. from the development of the cell culture under the influence of certain factors, to the efficacy of medicine or to a toxic effect of substances (e.g. in cosmetics), the reproducible cultivation and the standardized operation of the cell culture has a particular significance for the mentioned fields of application.
The possibility of standardizing the cell culture techniques is, due to its high dependency on manual (and thus user-dependent and not reproducible) methods and the resulting high efforts, at present not sufficient for the use of this culture in applications, such as stem-cell research, screening or tissue engineering.
A prerequisite for this are rather standardized cell culture methods, as only these permit a comparison between the substances and their active principles over years.
Correspondingly, the following individual problems exist:
The adaptation of the cell culturing conditions is performed, in existing solutions, on the basis of fixed protocols and, as a consequence, inflexible procedures in the cell cultivation process, these protocols being based on the manual cultivation by the laboratory personnel and their experience as well as the subjective perception of the operator and the established daily routine in the cell culture laboratory.
The control and documentation of the cell condition is normally effected only at an interval of hours or days as thus less efforts are involved. This results in long control intervals. In the cultivation of sensible cells, such as in particular stem cells, it is, however, often necessary to control the cells at shorter intervals.
Cell cultures are often used in pharmaceutical and biotechnological industries. In particular for ethical and economical reasons, animal experiments have been increasingly replaced by cell culture systems and cell culture technologies in recent years. For example, the European Union and the OECD also recommend the replacement of animal experiments by cell tests without animal experiments for testing medicine, chemicals and cosmetics. The employment of cell cultures in the search for new active principles and active agents in the pharmaceutical and pesticide sectors has therefore meanwhile become inevitable.
Moreover, a number of in-vitro test methods in the field of toxicology has been increasingly adopted in regulatory proceedings. Thus, in the pharmaceutical and pesticide sectors, too, the use of cell cultures is no longer dispensed with.
However, a significance of the data obtained with cell cultures is at present still limited by the existing analysis technique. Moreover, common manual methods in cell cultivation are time consuming and costly, and their results moreover highly depend on the user.
Due to this fact, initiatives have been started recently for defining minimum requirements on cell and tissue cultures and for thus ensuring the significance, comparability and reproducibility of in-vitro works. As a result, analogously to the Good Laboratory Practice (GLP) directives, principles for cell culture works have been passed with the designation Good Cell Culture Practice (GCCP).
These directives, however, have no influence on the fact that today, as a rule, cell cultures are still cultivated employing a lot of laboratory personnel. The cells are, as a rule, cultivated in Petri dishes, micro-titre plates or in cell culture bottles. In most cases, the steps necessary for cell cultivation, such as initial inoculation of the cells, change of the medium or passage of the cells, are performed merely manually. Moreover, the monitoring of the cells is discontinuous.
Changes of the pH value of the medium are observed by added color indicators.
The medium is changed at regular intervals, depending on the growth rate of the respective cells of the cell cultures (by manual control after removal from an incubator) to ensure sufficient supply of the cells with nutrients.
As one can draw conclusions from the results of cell cultivation, i.e. from the development of the cell culture under the influence of certain factors, to the efficacy of medicine or to a toxic effect of substances (e.g. in cosmetics), the reproducible cultivation and the standardized operation of the cell culture has a particular significance for the mentioned fields of application.
The possibility of standardizing the cell culture techniques is, due to its high dependency on manual (and thus user-dependent and not reproducible) methods and the resulting high efforts, at present not sufficient for the use of this culture in applications, such as stem-cell research, screening or tissue engineering.
A prerequisite for this are rather standardized cell culture methods, as only these permit a comparison between the substances and their active principles over years.
Correspondingly, the following individual problems exist:
The adaptation of the cell culturing conditions is performed, in existing solutions, on the basis of fixed protocols and, as a consequence, inflexible procedures in the cell cultivation process, these protocols being based on the manual cultivation by the laboratory personnel and their experience as well as the subjective perception of the operator and the established daily routine in the cell culture laboratory.
The control and documentation of the cell condition is normally effected only at an interval of hours or days as thus less efforts are involved. This results in long control intervals. In the cultivation of sensible cells, such as in particular stem cells, it is, however, often necessary to control the cells at shorter intervals.
In the characterization of the cells, the selection of the examined areas is made subjectively and arbitrarily by the operator. Here, even the assistance by software for the optical analysis is insofar manual and not reproducible.
The manual change of the cell culture media involves individual variations in time and thus variations in the supply for the cells. Equally, this results in stress for the cells, for example due to undefined flows.
In the characterization of the cells by the operator, the cells are moreover influenced due to great variations of the cell culturing conditions, for example changes of the ambient temperature of>IOK, that means, the culturing conditions are interrupted.
Their impact on cell development is not predictable.
Furthermore, there is a risk of contamination by a plurality of manual processes as well as by automation solutions of poor hygienic quality.
Another disadvantage is that the decision of the next step in the cell cultivation process is made according to a subjective assessment (for example depending on the operator, the weekday or time of day, or on imaging settings of the microscope) of the cell culture (for example evaluations, such as "degree of confluence approx. 70 or 80%", "medium approx.
consumed", or "cell during passage now approximately all detached") and according to the planning of the cell cultivation personnel or their work schedule (it is, for example, considered to be sufficient to passage or change the medium "tomorrow" or "on Monday after the weekend", or "after the holidays", and the personnel/the operator therefore does not have to perform the cell cultivation process outside his/her regular working hours).
In systems known on the market, cell culture chambers are used as self-sufficient system components besides automation systems, where the automation systems are, for example, exclusively usable for the passage or the medium change. These automation systems are no integral part of the cell culture process as the available systems, e.g. cell sowing, washing steps, medium change and cell harvest, are performed independently. Depending on the system, further steps, such as transfection or determination of the cell number, can be also carried out.
, ~ ' =
The manual change of the cell culture media involves individual variations in time and thus variations in the supply for the cells. Equally, this results in stress for the cells, for example due to undefined flows.
In the characterization of the cells by the operator, the cells are moreover influenced due to great variations of the cell culturing conditions, for example changes of the ambient temperature of>IOK, that means, the culturing conditions are interrupted.
Their impact on cell development is not predictable.
Furthermore, there is a risk of contamination by a plurality of manual processes as well as by automation solutions of poor hygienic quality.
Another disadvantage is that the decision of the next step in the cell cultivation process is made according to a subjective assessment (for example depending on the operator, the weekday or time of day, or on imaging settings of the microscope) of the cell culture (for example evaluations, such as "degree of confluence approx. 70 or 80%", "medium approx.
consumed", or "cell during passage now approximately all detached") and according to the planning of the cell cultivation personnel or their work schedule (it is, for example, considered to be sufficient to passage or change the medium "tomorrow" or "on Monday after the weekend", or "after the holidays", and the personnel/the operator therefore does not have to perform the cell cultivation process outside his/her regular working hours).
In systems known on the market, cell culture chambers are used as self-sufficient system components besides automation systems, where the automation systems are, for example, exclusively usable for the passage or the medium change. These automation systems are no integral part of the cell culture process as the available systems, e.g. cell sowing, washing steps, medium change and cell harvest, are performed independently. Depending on the system, further steps, such as transfection or determination of the cell number, can be also carried out.
, ~ ' =
In the characterization of cells by biochemical-biomolecular methods, at present one can only obtain snap-shots as the cells are often destroyed by the representation methods. The corresponding measuring methods are time consuming and not suited for monitoring cells over a relatively long period. A continued and consistent control and monitoring of the cells is not possible with the existing techniques.
The culturing conditions for the cells are often shaped by experience, such as, for example, by the intervals at which the culture medium is changed. This renders a possible standardization of the cell cultures difficult.
In contrast, systems which are capable of performing a regular monitoring of the cells at short intervals would be advantageous. Moreover, on the basis of the acquired data, the cultivation conditions would have to be readjusted if necessary.
Commercially available image analysis software for the analysis of cell microscopy images is either tuned to very special tasks (e.g. counting colored cells in suspensions), or it requires very good imaging skills of the user to exactly adapt the algorithms and parameters to the respective analysis task. In the image analysis of cell cultures, such an adaptation is absolutely necessary as different cell types can have very different appearances, and even cells of one type greatly vary depending on the cultivation stages. However, users in biology often do not have the required technical background for correspondingly adjusting the software.
Starting from the above statements, it is an object of the present invention to provide a method of cultivating a cell culture in an automated cell culture system and an automated cell culture system, wherein the automation is part of the cell culture process, to obtain optimized and standardized cell culturing conditions.
The object according to the invention is achieved with respect to the method aspect by a method of cultivating a cell culture in an automated cell culture system, wherein data concerning the condition of cells are acquired in the cell culture and at least one culturing condition is regulated in accordance with the acquired condition of the cells in the cell culture, and/or at least one operating step determined to be necessary in the cell cultivation process is initiated or carried out.
With respect to the apparatus aspect, the object according to the invention is achieved by an automated cell culture system with at least one actoric element for handling a cell culture, at least one sensory element for acquiring data concerning the condition of the cells in the cell culture, an apparatus for evaluating the data concerning the condition of the cells, and an apparatus for regulating cell culturing conditions and/or for initiating and/or carrying out further operating steps in the cultivation.
In the method according to the invention and the cell culture system according to the invention, optimized and standardized cell culturing conditions are achieved in that the culture is held in an automated cell culture system which acquires data (concerning the condition) of the cell and moreover, depending on their condition, regulates the cell culturing conditions (preferably directly) upon the acquisition of the condition of the cell culture and/or initiates/triggers necessary operating steps in the cell cultivation. Thereby, the influence of the human operator is reduced and replaced by a more precise automatic solution.
Moreover, a reproducible procedure is implemented which considers the natural variations of the living cell cultures.
Preferred embodiments of the method according to the invention and the automated cell culture system according to the invention are subject of the depending claims.
The present invention will be illustrated below with reference to preferred embodiments in connection with the corresponding drawing in which an optical arrangement for obtaining quantifiable spatial information of the cells and cell cultures is shown.
A preferred embodiment of the automated cell culture system comprises as actoric elements an automated handling unit for transporting cell culture vessels (for example cell culture bottles or micro-titre plates), an automated handling apparatus for liquids (for example the cell suspensions, fluorescent dyes, and nutrient media), and a storage for temporarily not used cell culture vessels.
The present automated cell culture system moreover comprises a conditioning unit for preparing the exterior cell culturing conditions, in particular a conditioned vertical circulation air unit with defined environmental conditions.
The present embodiment furthermore comprises a unit for generating a laminar flow, a medium supply, devices for the sterilization of surfaces, and a centrifuge. By a corresponding design of the surfaces as well as the choice of the material, decontamination properties are achieved by which the risk of cross-contamination is minimized. These include, for example, the prevention of unsealed threaded joints, of dead spaces or plane construction surfaces which would hinder the drainage of optional rinsing liquids or production residues.
Moreover, the condition of the cells in the cell culture in the present embodiment of the automated cell culture system is acquired with one or several devices for obtaining information from the culture. For this, an optical image acquisition and/or electrical/electrochemical sensors are provided (as sensory elements).
For example, an optical examination of the cell culture with various imaging microscopic methods, such as transmitted light, phase contrast or differential interference contrast, "DIC", are possible.
Possibly, an imaging fluorescent optic is added by which the spatial distribution of fluorescent dyes in the cell culture is determined.
These imaging methods are connected to an automatic image analysis which is suited for drawing conclusions to the condition of individual cells or the complete culture. Here, the spatial structure of the cells and cell cultures provides interesting additional information.
For obtaining such quantifiable information about a spatial structure of the cells and cell cultures, the conventional methods, such as phase contrast and differential interference contrast (DIC), are, however, not suited. These visually give an impression of the third dimension (in the observation direction, z-direction in the figure). This information, however, is not in all cases a quantitative information that can be further processed in an image analysis software. In particular, the images which are obtained with the mentioned conventional contrast method highly depend on the adjustment of the optics.
Objective, quantifiable information, however, is obtained by an interferometric method in which, in addition to the microscopic beam path (measurement beam path), an additional reference beam path is added.
In the (only) figure, such an optical arrangement for obtaining quantifiable spatial information about the cells and cell cultures is shown.
Here, a beam of light L is generated by a light source 1 and passed over a polarization filter 2.
The polarized beam with a wavelength of V2 is divided into two partial beams L1, L2 by means of a beam splitter 3. The partial beam LI, which forms the reference beam, passes through an assembly of a telescope and a spatial filter (designated with reference numeral 4).
The partial beam L2, which forms the measurement beam, is passed to sample P
via a deflection means 5 and a further polarization filter 6. After the passage through the sample P, the measurement beam L2 passes through a microscope objective 7. The measurement beam exiting from the microscope objective 7 is superposed by the reference beam L1 on an evaluation optics 8 (in particular a CCD field).
The beam division to the beam parts L1 and L2 is uniform in the present case, i.e. the intensity L 1 to L2 is 50% to 50%.
As can be seen in the (only) figure, the reference beam L 1 is superposed by the measurement beam L2 exiting from the objective. The interference pattern formed on the optical evaluation means 8 contains the information about the phase shift of the light at any location in the x-y-axis on the sample (in the figure, a Cartesian system of coordinates is shown). The optical phase is equivalent to the product of the thickness and the index of refraction of the observed object P. Assuming that the indices of refraction within the cells are homogenous and identical from cell to cell, one can calculate the thickness of the cells at any location in the x-y plane therefrom.
For definitely determining the phase, in the present embodiment, a component (telescope +
spatial filter) for the defined phase shift of the reference beam is included.
From several images which have been taken with various reference phases, a clear difference is made between phase and intensity information. At the same time, one obtains a phase value that is definite within a range of 27r. Beyond an interference order (phase change of 2n), the phase can be corrected by means of well-known "unwrapping" techniques.
Apart from the above-mentioned imaging methods, moreover optical methods are important by which the optical transmission or the fluorescence can be averaged and acquired over relatively large areas of the culture. With this, one can e.g. acquire the coloration of indicators which are added to the culture medium. By this, methods for averaging the cell properties can be made available.
Furthermore, electrical or electrochemical measuring methods can be employed by which, for instance, the local temperature, the pH value or ion concentrations are determined.
To be able to automatically determine the condition of the cell culture, software for the evaluation of image data and sensor signals and for controlling the complete course of the cell cultivation are provided. With this software, the image data of the cultivated cells acquired by the system are processed via an image analysis. The same supplies quantitative analysis results, such as for example cell density, number of dead cells, number of mitosis and number of morphologically modified cells, which give an image of the culture which can result, together with its history, i.e. data acquired earlier from this culture, and together with the protocols for the cultivation of the special culture, in a very exact evaluation of the present condition of the cell culture. This very exact evaluation of the condition of the cell culture is a solid basis for the determination of a possible intervention in the culturing conditions and/or for the initiation of an operating step in the cultivation process. This intervention can then be effected directly by the system, or it can lead to a message to the exterior.
In this case, the system can give a message, possibly with a suggestion for an action, which is then triggered by the operator.
The processing of the cell images and the sensor signals is designed as trainable software.
Thus, the system can be easily adapted to modified tasks (such as other cell cultures or modified basic conditions). The user presets, e.g. by means of images, a classification of cell types or of defined conditions of the culture, which are subsequently identified by the image processing during the cultivation.
In particular, here the image analysis software for the detection of relevant image structures, such as in particular healthy cells, shattered cells, detached cells, can be trained by the user.
To this end, in a training phase, examples of the structures to be identified are marked in some images. The image analysis software calculates a set of features (intensity, morphological features, textures) for these images and determines those features which characterize the selected examples, and in particular criteria which discriminate the structures searched for from other structures in the feature space. After the termination of the training phase, these calculated parameters can be used to automatically identify the desired structures in further images.
Apart from the automatic data acquisition and regulation, the documentation (e.g. the regulation parameter, the measured values of the regulation, the condition of the cell culture or the medium at different points of time, the measured values of the cultivation parameters and the condition of the cell culture on the basis of which a regulation or an operating step has been performed) is very important. In this way, the history of the culture and cultivation can be traced. Thus, a time controlled, more precise condition description is provided, and in case of a not optimal result, troubleshooting is facilitated.
In a first example of use of the present automated cell culture system, for an optimal cell differentiation, the composition of the differentiation medium is adapted to the degree of differentiation of the cell culture. For this, image data of the cell culture are continuously taken and evaluated by means of the evaluation software. In case of certain conditions of the cell which are identified by the software either the medium present in the cell culture vessel is automatically replaced by a new medium of a different composition, or growth factors are added to the present medium. Preferably, for the image analysis, the above described trainable software is used. The condition of the cell culture is acquired, for example, by means of morphological features or by means of minimally invasive marking substances. A
training of the image analysis software can be made in preliminary tests by experienced personnel or else by means of the invasive marking substances.
In a second example of use, the replacement of the culture medium is made in response to the cell function. In this case, a certain cell function, e.g. a contraction rate in case of cardiomyocytes, is quasi continuously observed. As soon as the parameters of the selected cell function leave a previously defined range, a replacement of the cell culture medium is caused.
In a third example of use, the cultivation of a first cell type is assisted by means of a further cell type (feeder cells). If in the mixed cultivation with these two cell types, the first cell type is present in a sufficient amount, the present culture medium is automatically replaced by a culture medium, which leads to the destruction of the second cell type. When all cells of the second type are dead, the culture medium is automatically replaced by normal medium.
In a fourth example of use, an operating step is initiated on the basis of a condition of the cell culture, such as a passage of the cell culture, when an intended degree of coverage/confluence is achieved, to further cell culture bottles or other cell containers, dispensing the cells in micro-titre plate wells, or individual cell withdrawal/handling/further processing.
In a fifth example of use, cells are cultivated in containers (e.g. Petri dishes, MTP, cell culture bottles), test substances (e.g. in case of active agent tests or toxicity tests) are added, and the cell culture in its property (e.g. growth rate, morphology, nucleus-plasma ratio, type of growth, such as for example criss-cross growth, formation of the cell function, cell counting in certain areas, for example colony center/colony edge) is documented in a time controlled manner, and a suggestion for the classification (e.g. normal, modified, transformed, cell number in the area, medium nucleus size, medium cytoplasm area, medium number of neutrites, etc.) is made to the system.
A sixth example of use relates to the optimization of the cell culture process in primary cell cultures, e.g. ceratinocytes from different biopsies, and/or the maintenance of the cell culture without further differentiating out.
A seventh example of use relates to an autonomous cultivation process of a robust routine cell culture (e.g. HeLa, HEK, COS), including change of medium, passage, sowing based on the optical and/or electrical/electrochemical sensory mechanism (e.g. for screening experiments with cells of the same quality within one cell culture) independent of regular working times of the laboratory personnel (e.g. over night, over the weekend, over holidays).
An eighth example of use relates to the standardization of a cell culture (definition of the process parameters for the cell cultivation, e.g. point of time for passage or change of medium, seeding density, planting efficiency (how many cells grow?)) with the present automated cell culture system on the basis of the documentation already illustrated above, before it is transferred to a system with rigid protocols or the cultivation is established in the bioreactor/fermenter.
In a ninth example of use, certain areas which have formed during the cultivation and comprise characteristic features are withdrawn from a cell culture. As soon as these areas have formed, this is detected by means of an image analysis, and the cells of the areas are automatically transferred to a new culture vessel by means of a pipette.
In a tenth example of use, aliquots from a cell suspension with a very low cell concentration are placed into wells of a micro-titre plate. In the regulated cell culture system, only cells of those wells are processed further in which there initially was exactly one cell.
From the above description, one can take in particular an automated cell culture system, consisting of actoric elements (for handling cell culture vessels, cell suspensions, fluorescent dyes and nutrient media), a conditioned vertical circulation air unit with defined environmental conditions, sensory elements (optical image acquisition and/or electrical sensory mechanism), and software for the evaluation of image data and sensor signals and for controlling the complete process, where the culturing conditions are regulated and/or operating steps are initiated and/or carried out in the cultivation depending on the situation.
The image acquisition is in this case made, among others, with an interferometric method.
Here, in addition to the microscopic beam path, a reference beam is introduced which is superposed by the measurement beam which exits from the microscope objective.
The processing of the cell images and the sensor signals is designed as trainable software in the present case. Thus, the system can be easily adapted to modified tasks (other cell cultures, modified basic conditions).
The system components are designed with a highly hygienic quality and thus prevent the contamination of the product. This is in particular true for the design of the surface roughness of smaller than 20 m, corresponding sealing elements of PTFE or similar materials, as well as a corresponding constructive design.
The planning of the automated cell cultivation is made on the basis of the desired end time of the production. Starting from a point of time at which the cell lines are to be available, a backward planning is carried out stating when a change of the medium and passages are to be carried out.
The culturing conditions for the cells are often shaped by experience, such as, for example, by the intervals at which the culture medium is changed. This renders a possible standardization of the cell cultures difficult.
In contrast, systems which are capable of performing a regular monitoring of the cells at short intervals would be advantageous. Moreover, on the basis of the acquired data, the cultivation conditions would have to be readjusted if necessary.
Commercially available image analysis software for the analysis of cell microscopy images is either tuned to very special tasks (e.g. counting colored cells in suspensions), or it requires very good imaging skills of the user to exactly adapt the algorithms and parameters to the respective analysis task. In the image analysis of cell cultures, such an adaptation is absolutely necessary as different cell types can have very different appearances, and even cells of one type greatly vary depending on the cultivation stages. However, users in biology often do not have the required technical background for correspondingly adjusting the software.
Starting from the above statements, it is an object of the present invention to provide a method of cultivating a cell culture in an automated cell culture system and an automated cell culture system, wherein the automation is part of the cell culture process, to obtain optimized and standardized cell culturing conditions.
The object according to the invention is achieved with respect to the method aspect by a method of cultivating a cell culture in an automated cell culture system, wherein data concerning the condition of cells are acquired in the cell culture and at least one culturing condition is regulated in accordance with the acquired condition of the cells in the cell culture, and/or at least one operating step determined to be necessary in the cell cultivation process is initiated or carried out.
With respect to the apparatus aspect, the object according to the invention is achieved by an automated cell culture system with at least one actoric element for handling a cell culture, at least one sensory element for acquiring data concerning the condition of the cells in the cell culture, an apparatus for evaluating the data concerning the condition of the cells, and an apparatus for regulating cell culturing conditions and/or for initiating and/or carrying out further operating steps in the cultivation.
In the method according to the invention and the cell culture system according to the invention, optimized and standardized cell culturing conditions are achieved in that the culture is held in an automated cell culture system which acquires data (concerning the condition) of the cell and moreover, depending on their condition, regulates the cell culturing conditions (preferably directly) upon the acquisition of the condition of the cell culture and/or initiates/triggers necessary operating steps in the cell cultivation. Thereby, the influence of the human operator is reduced and replaced by a more precise automatic solution.
Moreover, a reproducible procedure is implemented which considers the natural variations of the living cell cultures.
Preferred embodiments of the method according to the invention and the automated cell culture system according to the invention are subject of the depending claims.
The present invention will be illustrated below with reference to preferred embodiments in connection with the corresponding drawing in which an optical arrangement for obtaining quantifiable spatial information of the cells and cell cultures is shown.
A preferred embodiment of the automated cell culture system comprises as actoric elements an automated handling unit for transporting cell culture vessels (for example cell culture bottles or micro-titre plates), an automated handling apparatus for liquids (for example the cell suspensions, fluorescent dyes, and nutrient media), and a storage for temporarily not used cell culture vessels.
The present automated cell culture system moreover comprises a conditioning unit for preparing the exterior cell culturing conditions, in particular a conditioned vertical circulation air unit with defined environmental conditions.
The present embodiment furthermore comprises a unit for generating a laminar flow, a medium supply, devices for the sterilization of surfaces, and a centrifuge. By a corresponding design of the surfaces as well as the choice of the material, decontamination properties are achieved by which the risk of cross-contamination is minimized. These include, for example, the prevention of unsealed threaded joints, of dead spaces or plane construction surfaces which would hinder the drainage of optional rinsing liquids or production residues.
Moreover, the condition of the cells in the cell culture in the present embodiment of the automated cell culture system is acquired with one or several devices for obtaining information from the culture. For this, an optical image acquisition and/or electrical/electrochemical sensors are provided (as sensory elements).
For example, an optical examination of the cell culture with various imaging microscopic methods, such as transmitted light, phase contrast or differential interference contrast, "DIC", are possible.
Possibly, an imaging fluorescent optic is added by which the spatial distribution of fluorescent dyes in the cell culture is determined.
These imaging methods are connected to an automatic image analysis which is suited for drawing conclusions to the condition of individual cells or the complete culture. Here, the spatial structure of the cells and cell cultures provides interesting additional information.
For obtaining such quantifiable information about a spatial structure of the cells and cell cultures, the conventional methods, such as phase contrast and differential interference contrast (DIC), are, however, not suited. These visually give an impression of the third dimension (in the observation direction, z-direction in the figure). This information, however, is not in all cases a quantitative information that can be further processed in an image analysis software. In particular, the images which are obtained with the mentioned conventional contrast method highly depend on the adjustment of the optics.
Objective, quantifiable information, however, is obtained by an interferometric method in which, in addition to the microscopic beam path (measurement beam path), an additional reference beam path is added.
In the (only) figure, such an optical arrangement for obtaining quantifiable spatial information about the cells and cell cultures is shown.
Here, a beam of light L is generated by a light source 1 and passed over a polarization filter 2.
The polarized beam with a wavelength of V2 is divided into two partial beams L1, L2 by means of a beam splitter 3. The partial beam LI, which forms the reference beam, passes through an assembly of a telescope and a spatial filter (designated with reference numeral 4).
The partial beam L2, which forms the measurement beam, is passed to sample P
via a deflection means 5 and a further polarization filter 6. After the passage through the sample P, the measurement beam L2 passes through a microscope objective 7. The measurement beam exiting from the microscope objective 7 is superposed by the reference beam L1 on an evaluation optics 8 (in particular a CCD field).
The beam division to the beam parts L1 and L2 is uniform in the present case, i.e. the intensity L 1 to L2 is 50% to 50%.
As can be seen in the (only) figure, the reference beam L 1 is superposed by the measurement beam L2 exiting from the objective. The interference pattern formed on the optical evaluation means 8 contains the information about the phase shift of the light at any location in the x-y-axis on the sample (in the figure, a Cartesian system of coordinates is shown). The optical phase is equivalent to the product of the thickness and the index of refraction of the observed object P. Assuming that the indices of refraction within the cells are homogenous and identical from cell to cell, one can calculate the thickness of the cells at any location in the x-y plane therefrom.
For definitely determining the phase, in the present embodiment, a component (telescope +
spatial filter) for the defined phase shift of the reference beam is included.
From several images which have been taken with various reference phases, a clear difference is made between phase and intensity information. At the same time, one obtains a phase value that is definite within a range of 27r. Beyond an interference order (phase change of 2n), the phase can be corrected by means of well-known "unwrapping" techniques.
Apart from the above-mentioned imaging methods, moreover optical methods are important by which the optical transmission or the fluorescence can be averaged and acquired over relatively large areas of the culture. With this, one can e.g. acquire the coloration of indicators which are added to the culture medium. By this, methods for averaging the cell properties can be made available.
Furthermore, electrical or electrochemical measuring methods can be employed by which, for instance, the local temperature, the pH value or ion concentrations are determined.
To be able to automatically determine the condition of the cell culture, software for the evaluation of image data and sensor signals and for controlling the complete course of the cell cultivation are provided. With this software, the image data of the cultivated cells acquired by the system are processed via an image analysis. The same supplies quantitative analysis results, such as for example cell density, number of dead cells, number of mitosis and number of morphologically modified cells, which give an image of the culture which can result, together with its history, i.e. data acquired earlier from this culture, and together with the protocols for the cultivation of the special culture, in a very exact evaluation of the present condition of the cell culture. This very exact evaluation of the condition of the cell culture is a solid basis for the determination of a possible intervention in the culturing conditions and/or for the initiation of an operating step in the cultivation process. This intervention can then be effected directly by the system, or it can lead to a message to the exterior.
In this case, the system can give a message, possibly with a suggestion for an action, which is then triggered by the operator.
The processing of the cell images and the sensor signals is designed as trainable software.
Thus, the system can be easily adapted to modified tasks (such as other cell cultures or modified basic conditions). The user presets, e.g. by means of images, a classification of cell types or of defined conditions of the culture, which are subsequently identified by the image processing during the cultivation.
In particular, here the image analysis software for the detection of relevant image structures, such as in particular healthy cells, shattered cells, detached cells, can be trained by the user.
To this end, in a training phase, examples of the structures to be identified are marked in some images. The image analysis software calculates a set of features (intensity, morphological features, textures) for these images and determines those features which characterize the selected examples, and in particular criteria which discriminate the structures searched for from other structures in the feature space. After the termination of the training phase, these calculated parameters can be used to automatically identify the desired structures in further images.
Apart from the automatic data acquisition and regulation, the documentation (e.g. the regulation parameter, the measured values of the regulation, the condition of the cell culture or the medium at different points of time, the measured values of the cultivation parameters and the condition of the cell culture on the basis of which a regulation or an operating step has been performed) is very important. In this way, the history of the culture and cultivation can be traced. Thus, a time controlled, more precise condition description is provided, and in case of a not optimal result, troubleshooting is facilitated.
In a first example of use of the present automated cell culture system, for an optimal cell differentiation, the composition of the differentiation medium is adapted to the degree of differentiation of the cell culture. For this, image data of the cell culture are continuously taken and evaluated by means of the evaluation software. In case of certain conditions of the cell which are identified by the software either the medium present in the cell culture vessel is automatically replaced by a new medium of a different composition, or growth factors are added to the present medium. Preferably, for the image analysis, the above described trainable software is used. The condition of the cell culture is acquired, for example, by means of morphological features or by means of minimally invasive marking substances. A
training of the image analysis software can be made in preliminary tests by experienced personnel or else by means of the invasive marking substances.
In a second example of use, the replacement of the culture medium is made in response to the cell function. In this case, a certain cell function, e.g. a contraction rate in case of cardiomyocytes, is quasi continuously observed. As soon as the parameters of the selected cell function leave a previously defined range, a replacement of the cell culture medium is caused.
In a third example of use, the cultivation of a first cell type is assisted by means of a further cell type (feeder cells). If in the mixed cultivation with these two cell types, the first cell type is present in a sufficient amount, the present culture medium is automatically replaced by a culture medium, which leads to the destruction of the second cell type. When all cells of the second type are dead, the culture medium is automatically replaced by normal medium.
In a fourth example of use, an operating step is initiated on the basis of a condition of the cell culture, such as a passage of the cell culture, when an intended degree of coverage/confluence is achieved, to further cell culture bottles or other cell containers, dispensing the cells in micro-titre plate wells, or individual cell withdrawal/handling/further processing.
In a fifth example of use, cells are cultivated in containers (e.g. Petri dishes, MTP, cell culture bottles), test substances (e.g. in case of active agent tests or toxicity tests) are added, and the cell culture in its property (e.g. growth rate, morphology, nucleus-plasma ratio, type of growth, such as for example criss-cross growth, formation of the cell function, cell counting in certain areas, for example colony center/colony edge) is documented in a time controlled manner, and a suggestion for the classification (e.g. normal, modified, transformed, cell number in the area, medium nucleus size, medium cytoplasm area, medium number of neutrites, etc.) is made to the system.
A sixth example of use relates to the optimization of the cell culture process in primary cell cultures, e.g. ceratinocytes from different biopsies, and/or the maintenance of the cell culture without further differentiating out.
A seventh example of use relates to an autonomous cultivation process of a robust routine cell culture (e.g. HeLa, HEK, COS), including change of medium, passage, sowing based on the optical and/or electrical/electrochemical sensory mechanism (e.g. for screening experiments with cells of the same quality within one cell culture) independent of regular working times of the laboratory personnel (e.g. over night, over the weekend, over holidays).
An eighth example of use relates to the standardization of a cell culture (definition of the process parameters for the cell cultivation, e.g. point of time for passage or change of medium, seeding density, planting efficiency (how many cells grow?)) with the present automated cell culture system on the basis of the documentation already illustrated above, before it is transferred to a system with rigid protocols or the cultivation is established in the bioreactor/fermenter.
In a ninth example of use, certain areas which have formed during the cultivation and comprise characteristic features are withdrawn from a cell culture. As soon as these areas have formed, this is detected by means of an image analysis, and the cells of the areas are automatically transferred to a new culture vessel by means of a pipette.
In a tenth example of use, aliquots from a cell suspension with a very low cell concentration are placed into wells of a micro-titre plate. In the regulated cell culture system, only cells of those wells are processed further in which there initially was exactly one cell.
From the above description, one can take in particular an automated cell culture system, consisting of actoric elements (for handling cell culture vessels, cell suspensions, fluorescent dyes and nutrient media), a conditioned vertical circulation air unit with defined environmental conditions, sensory elements (optical image acquisition and/or electrical sensory mechanism), and software for the evaluation of image data and sensor signals and for controlling the complete process, where the culturing conditions are regulated and/or operating steps are initiated and/or carried out in the cultivation depending on the situation.
The image acquisition is in this case made, among others, with an interferometric method.
Here, in addition to the microscopic beam path, a reference beam is introduced which is superposed by the measurement beam which exits from the microscope objective.
The processing of the cell images and the sensor signals is designed as trainable software in the present case. Thus, the system can be easily adapted to modified tasks (other cell cultures, modified basic conditions).
The system components are designed with a highly hygienic quality and thus prevent the contamination of the product. This is in particular true for the design of the surface roughness of smaller than 20 m, corresponding sealing elements of PTFE or similar materials, as well as a corresponding constructive design.
The planning of the automated cell cultivation is made on the basis of the desired end time of the production. Starting from a point of time at which the cell lines are to be available, a backward planning is carried out stating when a change of the medium and passages are to be carried out.
Claims (20)
1. Method of cultivating a cell culture in an automated cell culture system, wherein data concerning the condition of cells are acquired in the cell culture and at least one culturing condition is regulated in accordance with the acquired condition of the cells in the cell culture, and/or at least one operating step determined to be necessary in the cell cultivation process is initiated or carried out.
2. Method according to claim 1, characterized in that for the determination of the data concerning the condition, at least one property of at least a part of the cells in the cell culture is acquired with an imaging method, and a classification of the cells is performed on the basis of the acquired property by an evaluation program.
3. Method according to claim 2, characterized in that for the regulation of the culturing conditions, at least one process parameter for the cell cultivation corresponding to the classification performed by the evaluation program is influenced by a regulation program for optimizing the cell culturing process.
4. Automated cell culture system with at least one actoric element for handling a cell culture, at least one sensory element for acquiring data concerning the condition of the cells in the cell culture, an apparatus for evaluating the data concerning the condition of the cells, and an apparatus for regulating cell culturing conditions and/or for initiating and/or carrying out further operating steps in the cultivation.
5. Automated cell culture system according to claim 4, characterized in that the actoric element for handling the cell culture vessels, the cell suspensions, the fluorescent dyes and/or the nutrient media is provided.
6. Automated cell culture system according to claim 4 or 5, characterized by a conditioned vertical circulation air unit with defined environmental conditions.
7. Automated cell culture system according to one of claims 4 to 6, characterized in that, as sensory elements, an optical image acquisition for the two- and/or three-dimensional representation of the cells and/or an electrical or electrochemical sensory mechanism are provided.
8. Automated cell culture system according to claim 7, characterized in that, as optical image acquisition, an imaging microscopic method, in particular transmitted light, phase contrast or differential interference contrast, are provided.
9. Automated cell culture system according to claim 7 or 8, characterized in that, as optical image acquisition, an interferometric method is provided in which, in addition to a microscopic beam path, a reference beam is provided, wherein the microscopic beam path as measurement beam can be superposed by the reference beam.
10. Automated cell culture system according to one of claims 7 to 9, characterized in that, as optical image acquisition, imaging fluorescent optics is provided by which a spatial distribution of fluorescent dyes in the cell culture can be determined.
11. Automated cell culture system according to one of claims 7 to 10, characterized by an apparatus for averaging the data acquired with the optical image acquisition over relatively larges areas of the cell culture, in particular an apparatus for the determination of the optical transmission or fluorescence.
12. Automated cell culture system according to one of claims 4 to 11, characterized by electrical and/or electrochemical sensors for the determination of process conditions, in particular for the determination of a local temperature, a pH value or an ion concentration.
13. Automated cell culture system according to one of claims 4 to 12, characterized in that the apparatuses for the evaluation of the data concerning the condition of the cells and for the regulation of cell culturing conditions and/or for the initiation and/or carrying out of further operating steps is designed as a software program for the evaluation of image data and sensor signals and for the control of the whole process.
14. Automated cell culture system according to claim 13, characterized in that the user can train an image analysis program part of the software program for the identification of
15 relevant image structures, in particular of healthy cells, cell debris and detached cells, wherein a user makes, with reference to images, a classification of cell types or of defined conditions of the culture, which can subsequently be identified by the image processing during the automatic cultivation.
15. Automated cell culture system according to claim 14, characterized in that in the training phase, some of the images can be marked as examples of structures to be identified, wherein the image analysis program part for these images calculates a set of features, comprising in particular intensity, morphological features and textures, and determines those features which characterize the selected examples, and determines criteria which discriminate the structures searched for from other structures in the feature space, and wherein, after the termination of the training phase, these calculated parameters are used to automatically identify the desired structures in further images.
15. Automated cell culture system according to claim 14, characterized in that in the training phase, some of the images can be marked as examples of structures to be identified, wherein the image analysis program part for these images calculates a set of features, comprising in particular intensity, morphological features and textures, and determines those features which characterize the selected examples, and determines criteria which discriminate the structures searched for from other structures in the feature space, and wherein, after the termination of the training phase, these calculated parameters are used to automatically identify the desired structures in further images.
16. Automated cell culture system according to one of claims 4 to 15, characterized by a storage means by which data and/or protocols acquired with the cell culture for the cultivation of cell cultures can be stored or called in.
17. Automated cell culture system according to claim 16, characterized in that the apparatus for regulating cell culturing conditions is provided for evaluating a present condition of the cell culture on the basis of the data acquired with the cell culture and stored protocols of earlier cultivations for the cultivation of the present cell cultures, wherein this evaluation is the basis for a determination of a possible intervention in the culturing conditions.
18. Automated cell culture system according to one of claims 4 to 17, characterized in that the apparatus for the regulation of cell culturing conditions is provided for carrying out the intervention directly in the system with the automatic actoric elements for the handling and for the operation of the cell culture, and/or for giving a message to the exterior to the operators, in particular together with a suggestion for possible interventions.
19. Automated cell culture system according to one of claims 4 to 18, characterized in that a planning of the automated cell cultivation is made with reference to a desired end time of the production, wherein, starting from the point of time at which the cell line are to be available, a backward planning is made stating when a change of the medium and passages are to be performed.
20. Automated cell culture system according to one of claims 4 to 19, characterized in that the system components are designed with a high hygienic quality, wherein a surface roughness is smaller than 20 µm, and wherein in particular sealing elements are made of PTFE or similar materials.
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| DE102005021034.1 | 2005-05-06 | ||
| PCT/EP2006/003760 WO2006119860A1 (en) | 2005-05-06 | 2006-04-24 | Cell culture system and method of cultivating a cell culture |
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| CN106471508A (en) * | 2014-06-20 | 2017-03-01 | 康涅狄格儿童医疗中心 | Automated cell culture system and corresponding method |
| WO2019241885A1 (en) * | 2018-06-19 | 2019-12-26 | Stemcell Technologies Canada Inc. | Systems, methods and apparatus for the automated culture of cells |
| EP4092376A4 (en) * | 2020-01-17 | 2023-06-21 | FUJIFILM Corporation | Information processing device, operation method for information processing device, and operation program for information processing device |
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| GB201004614D0 (en) | 2010-03-19 | 2010-05-05 | Ge Healthcare Uk Ltd | A system and method for automated extraction of multi-cellular physiological parameters |
| JP6291388B2 (en) * | 2014-09-12 | 2018-03-14 | 富士フイルム株式会社 | Cell culture evaluation system and method |
| WO2019050888A1 (en) * | 2017-09-07 | 2019-03-14 | Corning Incorporated | Optical cell culture monitoring and analyte measuring system |
| CN115862009B (en) * | 2023-02-24 | 2023-05-12 | 山东卫康生物医药科技有限公司 | Data processing method and system for precisely culturing ginseng stem cells |
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| US4179339A (en) * | 1977-03-02 | 1979-12-18 | Olympus Optical Company, Ltd. | Liquid feeder for automatic culture apparatus |
| GB9714347D0 (en) * | 1997-07-09 | 1997-09-10 | Oxoid Ltd | Image analysis systems and devices for use therewith |
| JP4402249B2 (en) * | 2000-03-31 | 2010-01-20 | 正仁 田谷 | Cell culture method, cell culture apparatus and recording medium |
| EP1205742B1 (en) * | 2000-09-29 | 2006-08-09 | Deutsches Krebsforschungszentrum | Automated classification of cellular structures |
| DE10128810B4 (en) * | 2001-06-18 | 2013-11-07 | Pan-Biotech Gmbh | Device for cultivating cells, in particular human or animal cells |
| US20050170491A1 (en) * | 2002-07-31 | 2005-08-04 | Mutsumi Takagi | Automatic culture apparatus for cell or tisse with biological origin |
| US20060275888A1 (en) * | 2003-04-09 | 2006-12-07 | Hiroki Hibino | Culture treatment apparatus and automatic culture apparatus |
| DE10329539A1 (en) * | 2003-06-30 | 2005-01-20 | Innovatis Ag | Method and device for characterizing a culture fluid |
| FI20040958A (en) * | 2004-07-09 | 2006-01-10 | Chip Man Technologies Oy | Method and apparatus for biological analysis |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106471508A (en) * | 2014-06-20 | 2017-03-01 | 康涅狄格儿童医疗中心 | Automated cell culture system and corresponding method |
| WO2019241885A1 (en) * | 2018-06-19 | 2019-12-26 | Stemcell Technologies Canada Inc. | Systems, methods and apparatus for the automated culture of cells |
| WO2020252560A1 (en) * | 2018-06-19 | 2020-12-24 | Stemcell Technologies Canada Inc. | Systems, methods and apparatus for adaptive passage of a culture of cells |
| CN112585257A (en) * | 2018-06-19 | 2021-03-30 | 加拿大干细胞科技公司 | System, method and device for automatic cell culture |
| EP4092376A4 (en) * | 2020-01-17 | 2023-06-21 | FUJIFILM Corporation | Information processing device, operation method for information processing device, and operation program for information processing device |
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| DE102005021034B4 (en) | 2012-09-13 |
| EP1877535B1 (en) | 2019-06-12 |
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| DE102005021034A1 (en) | 2006-11-16 |
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