CN114574366B - Cell culture apparatus and method - Google Patents

Abstract

The invention provides a cell culture device and a cell culture method, wherein the cell culture device comprises a culture unit, a conveying unit and a monitoring unit, the culture unit comprises a first culture container, and the monitoring unit comprises a light source, a photoelectric detector and an analysis module; the bottom of the first culture container is provided with an outer convex part, and the breathable film is arranged at the bottom end of the first culture container; the light source is arranged on the upper side of the outer convex part, and the photoelectric detector is arranged on the lower side of the outer convex part. The invention has the advantages of automation and the like.

Description

Cell culture device and method

Technical Field

The present invention relates to cell culture, and more particularly to a cell culture device and method.

Background Art

The environment for in vitro cell culture must also have certain physical and chemical properties to maintain normal physiological conditions required for cell survival and proliferation, including temperature, osmotic pressure, gas phase and pH. Traditional cell culture uses containers such as culture flasks and culture dishes. However, the gas exchange of culture flasks is mainly carried out through the breathable membrane on the bottle cap, which has a small area. If there is no breathable bottle cap, it is easy to contaminate by loosening the bottle cap. The gas exchange space of the culture dish is small and easily contaminated.

In addition, in the process of culturing cells, cell passaging is a routine operation. Passaging can expand the number of cells in culture, and also avoid contact inhibition caused by cells entering the stable period, or even a large number of cell deaths in the decay period. However, traditional cell passaging requires visual observation, which is highly subjective, and requires manual opening of culture bottles and culture dishes; it also requires deep pipetting for liquid manipulation, which poses a high risk of contamination.

Summary of the invention

In order to solve the deficiencies in the above-mentioned prior art solutions, the present invention provides a cell culture device.

The objective of the present invention is achieved through the following technical solutions:

A cell culture device, comprising a culture unit, a transport unit and a monitoring unit, wherein the culture unit comprises a first culture container, and the monitoring unit comprises a light source, a photodetector and an analysis module;

The bottom of the first culture container has an outer convex portion, and the air permeable membrane is arranged at the bottom end of the first culture container;

The light source is disposed on the upper side of the outer protrusion, and the photodetector is disposed on the lower side of the outer protrusion.

The object of the present invention is also to provide a cell culture method, which is achieved by the following technical solutions:

A cell culture method, wherein the cell culture method comprises:

The cells are cultured on a gas permeable membrane at the bottom of a first culture container, the bottom of the first culture container has an outer convex portion, and the gas permeable membrane is arranged at the bottom end of the first culture container;

The light emitted by the light source illuminates the cells on the upper side of the breathable membrane in the protrusion, and the photoelectric detector on the lower side of the breathable membrane receives the light signal, converts it into an electrical signal and sends it to the analysis module;

The analysis module obtains an image of the cells according to the electrical signals and obtains the cell culture status.

Compared with the prior art, the present invention has the following beneficial effects:

1. Automation;

By providing an outer protrusion at the bottom of the first culture container, the cell culture status can be monitored in real time, and the culture medium can be replaced, cells can be transferred to expand the culture volume, etc. according to the culture status. The whole process is automated, no manual operation is required, and the subjectivity of monitoring is avoided;

2. Accurate cell monitoring results;

By using image segmentation and parameter matrix construction, the grayscale value of each pixel in the image is corrected, so that the image illumination is balanced and the image restoration effect is improved;

In the segmentation of cell images, not only the image restoration method of this patent is used, but also double threshold segmentation and hole filling are combined to achieve accurate segmentation and improve the accuracy of cell monitoring;

3. Simple structure;

Multiple culture containers are arranged as required, but a monitoring unit is arranged only on the first culture container, and the culture conditions of other culture containers are obtained by using the culture conditions output by the monitoring unit, thereby reducing the structural complexity and cost of the two-way device.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure of the present invention will become easier to understand with reference to the accompanying drawings. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. In the drawings:

FIG1 is a schematic structural diagram of a cell culture device according to an embodiment of the present invention;

FIG2 is another schematic structural diagram of a cell culture device according to an embodiment of the present invention;

FIG. 3 is a schematic flow chart of the working mode of the analysis module according to an embodiment of the present invention.

DETAILED DESCRIPTION

Fig. 1-3 and the following description describe the optional embodiment of the present invention to teach those skilled in the art how to implement and reproduce the present invention. In order to explain the technical solution of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art will appreciate that variations or replacements derived from these embodiments will be within the scope of the present invention. Those skilled in the art will appreciate that the following features can be combined in various ways to form multiple variations of the present invention. Thus, the present invention is not limited to the following optional embodiments, but is only limited by the claims and their equivalents.

Embodiment 1:

FIG1 schematically shows a simplified structural diagram of a cell culture device according to Example 1 of the present invention. As shown in FIG1 , the cell culture device comprises:

A culture unit, the culture unit comprising a first culture container 11, the first culture container 11 is in a sealed state; the bottom of the first culture container 11 has an outer protrusion 12, and a gas permeable membrane 13 is arranged at the bottom end of the first culture container 11, that is, the bottom end of the outer protrusion 12 is the gas permeable membrane 13;

A conveying unit 21, comprising a pump and a pipeline, for conveying seed liquid or culture medium into the first culture container 11;

The monitoring unit includes a light source 61 , a photodetector 62 and an analysis module; the light source 61 is arranged on the upper side of the outer protrusion 12 , and the photodetector 62 is arranged on the lower side of the outer protrusion 12 .

In order to reduce biological toxicity, the breathable membrane 13 further includes protein, liposome and silica gel, and the mass ratio is 1:200-290:500-700. For example, the pore size of the silica gel is 100-200 angstroms and the particle size is 4-10 μm; the liposome includes lecithin.

In order to increase the number of cells cultured, the culture unit further includes a second culture container 31, and a gas permeable membrane 32 is arranged at the bottom of the second culture container 31; the first culture container 11 and the second culture container 31 are connected through a conveying unit 21.

In order to reduce the complexity of the structure, further, the monitoring unit and the outer protrusion 12 are not provided on the second culture container 31, and the cell culture status in the first culture container 11 obtained by the monitoring unit is used to understand the cell culture status in the second culture container 31, because the cells and culture medium in the two culture containers are the same.

The cell culture method of the embodiment of the present invention comprises:

The cells are cultured on a gas permeable membrane 13 at the bottom of a first culture container 11. The bottom of the first culture container 11 has an outer protrusion 12. The gas permeable membrane 13 is arranged at the bottom end of the first culture container 11.

The light emitted by the light source 61 illuminates the cells on the upper side of the gas permeable membrane 13 in the protrusion 12, and the photodetector 62 on the lower side of the gas permeable membrane 13 receives the light signal, converts it into an electrical signal and sends it to the analysis module;

The analysis module obtains an image of the cells according to the electrical signals and obtains the cell culture status.

In order to improve the accuracy of the cell culture status, further, as shown in FIG3 , the working method of the analysis module includes the following steps:

(A1) dividing the image of size M×N into a plurality of sub-blocks of size m×n, where M=m×s and N=n×t;

(A2) Obtain the average grayscale value μ of the image and the average grayscale value μ _cd of the c-th row and d-th column sub-block, thereby obtaining the ratio corresponding to the c-th row and d-th column sub-block

p(i,j) is the grayscale value of the pixel at coordinate (i,j) in the image;

(A3) Constructing the adjustment parameter matrix

(A4) For the neighborhood range Ω of size (2l+1)×(2l+1) centered on r _cd in the matrix R′, the correction value of the ratio r _cd is obtained (a, b) are the coordinate values of each element in the neighborhood range Ω, σ∈[5.5,7], l is a positive integer, such as 1, 2 or 3;

(A5) obtaining a grayscale value p(i,j) correction value p′(i,j)=p(i,j)×r′ _cd of the pixel point in the sub-block of the cth row and the dth column, thereby obtaining a restored image;

(A6) performing double threshold segmentation on the restored image to realize image binarization;

(A7) Filling the cell area to obtain the culture status of the cells, such as cell confluence.

In order to further repair the image, further, in step (A5), the average gray value μ′ of the repaired image is obtained, and the correction value of the pixel point of the repaired image is obtained.

In order to improve the segmentation effect, the morphological hole filling method is further used to fill the cell area, and then the area filtering method is used for processing.

In order to increase the number of cells cultured, further, during the cell culture process, part of the cells in the first culture container are transferred to the second culture container, and the cell culture status in the second culture container is obtained by using the cell culture status in the first culture container.

Embodiment 2:

Application examples of the cell culture device and method of the image according to Example 1 of the present invention.

In this application example, as shown in FIG1 , in the cell culture device, the culture unit is provided with only one culture container, namely, the first culture container 11, and the first culture container 11 is cylindrical; the gas permeable membrane 13 includes protein, liposome and silica gel, and the mass ratio is 1:240:600, the pore size of the silica gel is 150 angstroms, and the particle size is 7 μm; the liposome includes lecithin, such as egg yolk lecithin, soybean lecithin, artificial synthetic lecithin, sphingomyelin, etc.;

In the monitoring unit, an objective lens and a photodetector 62 are sequentially arranged on the lower side of the outer protrusion 12, and the photodetector 62 adopts a CCD;

The conveying unit 21 includes a first pipeline, a second pipeline and a third pipeline. One end of the first pipeline is connected to the seed liquid, the culture medium and the sterile liquid storage bag respectively by a switching module, and the other end is at the bottom of the first culture container 11. One end of the second pipeline is connected to the waste liquid bag, and the other end is in the first culture container 11, and the position is higher than the bottom end position of the first pipeline in the first culture container 11.

The cell culture method of the embodiment of the present invention, that is, the working method of the cell culture device of the present embodiment, is as follows:

By switching the switching module, the seed liquid is sent to the first culture container 11 through the first pipeline of the conveying unit 2, and the cells are cultured on the gas permeable membrane 13 at the bottom of the first culture container 11. The bottom of the first culture container 11 has an outer protrusion 12, and the gas permeable membrane 13 is arranged at the bottom end of the first culture container 11;

The light emitted by the light source 61 illuminates the cells on the upper side of the gas permeable membrane 13 in the protrusion 12, and the photodetector 62 on the lower side of the gas permeable membrane 13 receives the light signal, converts it into an electrical signal and sends it to the analysis module;

The analysis module obtains the image of the cells according to the electrical signals and obtains the cell culture status:

If the culture time is long and the medium needs to be replaced, part of the culture medium in the first culture container 11 needs to be extracted using the second pipe of the conveying unit 2; and then new culture medium needs to be injected into the first culture container 11 using the first pipe;

If the culture meets the requirements, the switching module is switched, and the cells in the first culture container 11 are sent to the sterile liquid storage bag through the first pipeline;

As shown in FIG3 , the working method of the analysis module includes the following steps:

(A1) dividing the image of size M×N into a plurality of sub-blocks of size m×n, where M=m×s and N=n×t;

(A2) Obtain the average grayscale value μ of the image and the average grayscale value μ _cd of the c-th row and d-th column sub-block, thereby obtaining the ratio corresponding to the c-th row and d-th column sub-block

p(i,j) is the grayscale value of the pixel at coordinate (i,j) in the image;

(A3) Constructing the adjustment parameter matrix

(A4) For the neighborhood range Ω of size (2l+1)×(2l+1) centered on r _cd in the matrix R′, the correction value of the ratio r _cd is obtained (a, b) are the coordinate values of each element in the neighborhood range Ω, σ∈[5.5,7], l is 1, 2 or 3;

(A5) obtaining a grayscale value p(i,j) correction value p′(i,j)=p(i,j)×r′ _cd of the pixel point in the c-th row and d-th column sub-block, thereby obtaining a preliminary restored image;

(A6) Obtain the average grayscale value μ′ of the image after preliminary restoration, and obtain the correction value of the pixel point of the restored image

Perform double threshold segmentation on the repaired image to realize image binarization, specifically:

First, the processed image is Gaussian filtered to eliminate some background noise. Then, according to the image characteristics, the image is divided into three categories: halo, background, and cells. Two optimal thresholds are selected to maximize the inter-class variance between the three categories. The image is classified based on the two optimal thresholds. Finally, the grayscale of the background and halo is set to 0, and the cell pixel is set to 255 to achieve image binarization.

A morphological hole filling method is used to fill part of the cell area, and at the same time, an area filtering method is used to filter out some fine impurities, and the segmented cell areas are statistically sorted; in this embodiment, the area filtering threshold is set to 50, and the connected domain area less than 50 will be filtered out, thereby obtaining the cell culture status, such as confluence.

Embodiment 3:

The application example of the cell culture device and method of the image according to Example 1 of the present invention is different from Example 2 in that:

1. In the cell culture device, as shown in FIG2 , a second culture container 31 is further included. A gas permeable membrane 32 is disposed at the bottom of the second culture container 31 . The gas permeable membrane 32 is the same as the gas permeable membrane 13 at the bottom of the first culture container 11 . The second culture container 31 is not provided with an outer protrusion 12 , that is, no monitoring unit is required.

The transport unit further includes more pipelines, such as using pipelines to connect the second culture container 31 to the culture medium, the waste liquid bag and the sterile liquid storage bag;

2. In the cell culture method, for the cell culture status output by the analysis module:

If the cell confluence in the first culture container 11 exceeds a threshold value, such as 85%, the conveying unit 2 is used to deliver part of the cells in the first culture container 11 to the second culture container 31, and the cell culture status in the second culture container 31 is obtained by using the cell culture status in the first culture container 11 output by the monitoring unit;

If the culture time is long and the medium needs to be replaced, the pipeline of the conveying unit 2 needs to be used to extract part of the culture medium in the first culture container 11 and the second culture container 31 respectively; and then the pipeline needs to be used to inject new culture medium into the first culture container 11 and the second culture container 31;

If the culture reaches the requirements, the cells in the first culture container 11 and the second culture container 31 are respectively sent to the sterile liquid storage bag through the pipeline.

Embodiment 4:

The application example of the cell culture device and method of the image according to Example 1 of the present invention is different from Example 3 in that:

The first culture container and the second culture container have the same structure, that is, the second culture container has the same outer protrusion as the first culture container.

Claims (4)

1. A cell culture method, the cell culture method comprising:

the method comprises the steps that cells are cultured on a breathable film at the bottom of a first culture container, wherein the bottom of the first culture container is provided with an outer convex part, and the breathable film is arranged at the bottom end of the first culture container;

the light emitted by the light source irradiates cells on the upper side of the ventilated membrane in the outer part, and the photoelectric detector on the lower side of the ventilated membrane receives the light signal, converts the light signal into an electric signal and sends the electric signal to the analysis module;

The analysis module obtains an image of the cells according to the electric signals and obtains cell culture conditions;

The working mode of the analysis module comprises the following steps:

(A1) Dividing the image of size m×n into a plurality of sub-blocks of size m×n, m=m×s, n=n×t;

(A2) Obtaining an average gray value mu of the image and average gray values mu _cd of the c-th row and the d-th column sub-blocks, thereby obtaining the ratio corresponding to the c-th row and the d-th column sub-blocks ，c=1,2…s，d=1,2…t；

，P (i, j) is the gray value of the pixel point at the coordinate (i, j) in the image;

(A3) Constructing an adjustment parameter matrix ；

(A4) For matrixIn which r _cd is the centerIs included in the neighborhood range omegaIndividual matrixTo obtain the correction value of the ratio r _cd ，，，Is a positive integer, (a, b) is the coordinate value of each element in the neighborhood range omega,；

(A5) Obtaining the correction value of the gray value p (i, j) of the pixel point in the sub-blocks of the c row and the d columnThereby obtaining a repaired image;

(A6) Performing double-threshold segmentation on the repaired image to realize binarization of the image;

(A7) The cell area is filled, and thus the culture condition of the cells is obtained.

2. The method according to claim 1, wherein in the step (A5), the average gray value of the restored image is obtainedObtaining a correction value of the repaired image pixel point，，。

3. The cell culture method according to claim 1, wherein the cell area is filled by a morphological hole filling method and then treated by an area filtering method.

4. The method according to claim 1, wherein a part of the cells in the first culture vessel is transferred to the second culture vessel during the cell culture, and the cell culture condition in the second culture vessel is obtained by using the cell culture condition in the first culture vessel.