Cell culture microcapsule preparation system and preparation method
By setting electrodes in the microcapsule preparation pipeline to measure impedance changes, and combining the analysis control unit to adjust the input flow, the problem of microcapsule size and wall thickness control is solved, and efficient and low-cost microcapsule production and quality control is achieved, and cells are improved Efficiency and accuracy of cultivation.
Patent Information
- Application Number
- CN202411461500.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2025-01-14
AI Technical Summary
The existing microcapsule preparation technology cannot accurately control the size and wall thickness of the microcapsule, resulting in low production efficiency, high cost and difficult quality control in cell culture, especially in the resistance detection method in acidified oil medium. Spotlight noise interference makes it difficult to achieve high-precision online detection.
By setting electrodes in the preparation pipeline, measuring impedance changes in the output pipeline, combining the analysis control unit to adjust the gel input flow and pressure, dynamically adjust the parameters of the microcapsules, and realizing preset values of microcapsules production. The electrode uses AC power supply and differential processing signals to improve detection accuracy.
Real-time adjustment of microcapsules size and wall thickness is achieved, ensuring efficient cultivation of cells in 3D culture reactors, reducing production costs, and improving detection accuracy through high-frequency alternating current measurement.
Smart Images
Figure CN119307346A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological culture equipment, and in particular to a cell culture microcapsule preparation system and method. Background Art
[0002] As cell drugs enter clinical applications, traditional 2D cell culture system cell factories are difficult to meet the needs of large-scale applications, with low production efficiency and high unit cost. Existing 3D culture reactors have higher specific surface area, high mass transfer efficiency, easy scale-up, and lower unit cost.
[0003] Then, when culturing cells in a 3D culture reactor, stirring and other operations can easily cause negative effects on the cells, such as damage. Through the microcapsule 3D cell culture method, cells can be encapsulated in microcapsules and placed in a culture reactor for culture, so that the cells can more realistically simulate the in vivo growth environment.
[0004] In the existing microcapsule production process, gel is introduced into the periphery at the same time as the cells are introduced, so that the cells are covered by the gel, and then the gel is cut off by acidified oil shock. The microcapsules are separated from the oil phase and sent to a culture reactor for culture.
[0005] However, the microcapsule production process cannot accurately control the size, wall thickness and other parameters of the microcapsules. Therefore, for the large-scale application of microcapsule cell culture, it is urgent to develop an online, accurate, low-cost and stable microcapsule detection technology to feedback control the microcapsule production parameters to achieve microcapsule quality control. However, the existing microcapsule online detection method using optical microscopy technology has problems such as large space occupation, low time resolution, and complex data processing. In addition, due to the close refractive index of the capsule shell and the core material, it is difficult to perform online analysis of the shell thickness by optical methods. Although electrical methods (especially resistance detection methods) have been reported for high-throughput detection of single cells, organoids or microcapsules, existing studies are all based on offline and in electrolyte solutions with good conductivity. Due to the low conductivity of acidified oil, it has a large impedance itself, which will introduce interference factors such as thermal noise and shot noise into the measurement results. Therefore, it also poses a challenge to establish an impedance-based microcapsule online detection method. Summary of the invention
[0006] The purpose of the present invention is to solve the above problems and provide a cell culture microcapsule preparation system and method. The impedance change in the pipeline when the microcapsules are output is measured by electrodes, and the size parameters of the microcapsules are measured to control the gel input flow rate so that the output microcapsules reach the predetermined parameters.
[0007] The technical solution adopted by the present invention is: A cell culture microcapsule preparation system is characterized in that it includes a preparation pipeline, a microcapsule outer layer input unit, a cell input unit and a microcapsule solidified oil input unit arranged at the front end of the preparation pipeline, and is used to prepare microcapsules. The rear end of the preparation pipeline is an output pipeline, and electrodes are arranged in the output pipeline. The electrodes are connected to an analysis control unit, and the analysis control unit obtains parameter information of the microcapsule according to the impedance change in the output pipeline detected by the electrodes. According to the parameter information of the microcapsule, the analysis control unit adjusts the input flow and pressure of the microcapsule outer layer input unit, the cell input unit and the microcapsule solidified oil input unit, and dynamically adjusts the parameter information of the microcapsule to a preset value.
[0008] Furthermore, the electrode includes a source electrode and at least one receiving electrode, the source electrode is connected to an AC power source, the AC power frequency is 100 kHz to 10 MHz, or a mixed signal of several different frequencies within the above frequency range.
[0009] Furthermore, there are two receiving electrodes, which are symmetrically arranged on both sides of the source electrode, and the analysis and control unit processes the signals on the two receiving electrodes differentially.
[0010] Furthermore, the parameter information of the microcapsule includes the external shape, size, wall thickness and number of cells in the microcapsule.
[0011] Furthermore, the arrangement direction of the electrodes forms an angle with the flow direction of the microcapsules in the output pipe.
[0012] Furthermore, the diameter of the microcapsule is 10 μm-1000 μm, and the electrode gap is 0.1 to 2 times the diameter of the microcapsule.
[0013] Furthermore, the outer layer of the microcapsule input by the microcapsule outer layer input unit is sodium alginate gel, the microcapsule solidifying oil input by the microcapsule solidifying oil input unit comprises a mixture of mineral oil, calcium chloride and a surfactant, and the sodium alginate is mixed with the calcium chloride in the microcapsule solidifying oil and then solidified to form microcapsules.
[0014] A method for preparing cell culture microcapsules, characterized in that it comprises the following steps: (1) The microcapsule outer layer input unit, the cell input unit and the microcapsule solidified oil input unit generate microcapsules in the preparation pipeline, and the microcapsules flow out through the output pipe of the preparation pipeline; (2) The electrodes on the output pipeline measure the fluid impedance signal in the output pipeline; (3) The analysis control unit obtains parameter information of the microcapsules in the current output pipeline according to the impedance signal; (4) adjusting the input flow and pressure of the microcapsule outer layer input unit, the cell input unit, and the microcapsule solidified oil input unit according to the comparison between the current microcapsule parameter information and the preset value; (5) Repeat steps (2)-(4) to dynamically adjust the parameter information of the microcapsule to reach the preset value.
[0015] Furthermore, in step (3), the parameter information of the microcapsule is the outer diameter and the wall thickness, the impedance signal is a plurality of sharp waves, each sharp wave represents a microcapsule, and the outer diameter and the wall thickness of the microcapsule are calculated according to each sharp wave signal.
[0016] Furthermore, in step (4), the input flow rate and pressure of the microcapsule outer layer input unit are in direct proportion to the outer diameter and wall thickness of the microcapsule, and the input flow rate and pressure of the microcapsule outer layer input unit are adjusted according to the comparison between the current outer diameter and wall thickness of the microcapsule and the preset values.
[0017] The beneficial effects of the present invention are: (1) Real-time adjustment of the gel input flow rate to make the parameters of the microcapsules reach the predetermined values, ensuring efficient cell culture in the 3D culture reactor; (2) The impedance change between electrodes is measured in the pipe solution by high-frequency alternating current, and the size parameters of the microcapsules are calculated; (3) Through the three-electrode method, the signals on the two receiving electrodes are differentially processed to improve the measurement accuracy. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Attached Figure 1 It is a schematic diagram of the structure of the preparation system of the present invention; Attached Figure 2 This is another schematic diagram of electrode arrangement; Attached Figure 3 It is the electrode detection impedance waveform; Attached Figure 4 It is a linear relationship diagram between the electric diameter and the microcapsule. DETAILED DESCRIPTION
[0019] The specific implementation methods of the cell culture microcapsule preparation system and preparation method of the present invention are described in detail below with reference to the accompanying drawings.
[0020] See attached Figure 1The cell culture microcapsule preparation system includes a preparation pipeline, a microcapsule outer layer input unit 1, a cell input unit 2 and a microcapsule solidified oil input unit 3 arranged at the front end of the preparation pipeline. During operation, cells, microcapsule outer layers and microcapsule solidified oil are periodically input in sequence. When the cells are input, they are coated by the microcapsule outer layer. When they are transported forward, the microcapsule solidified oil cuts them off to form microcapsules that continuously output the coated cells. The microcapsule outer layer input unit 1 inputs sodium alginate gel, which has gel properties, and coats the cells. At the same time, it has pores that pass into the culture medium to keep the cells growing.
[0021] The cells in the cell culture medium are input into the culture matrix and mixed. The culture matrix is composed of hyaluronic acid or matrix gel mixed with a certain amount of sodium alginate, and then the mixed solution is input into the microfluidic preparation pipeline. The microcapsule curing oil uses a mixed solution of mineral oil, surfactant, and calcium chloride. When calcium chloride comes into contact with sodium alginate, the sodium alginate is cured into a hydrogel to form microcapsules that encapsulate the cells.
[0022] The microcapsules are outputted in the output pipe 4 at the rear end of the preparation pipeline. Electrodes are arranged in the output pipe 4, and the electrodes are connected to the analysis control unit, which detects the impedance change in the output pipe 4 according to the electrodes. According to the analysis of experimental data, the applicant found that the impedance change outputted by the electrodes has a regular pattern with the parameter information of the microcapsules, such as the external shape, size, wall thickness, and number of cells in the microcapsules.
[0023] The following is an experimental analysis of the relationship between the diameter and wall thickness of the microcapsules and the electrode impedance.
[0024] According to the analysis of experimental data, there is a regularity between the impedance change in the output pipe 4 and the diameter and wall thickness of the microcapsule, and the diameter and wall thickness of the microcapsule can be obtained by calculation. The diameter and wall thickness of the microcapsule are linearly related to the input flow rate of the microcapsule outer layer input unit 1. Therefore, the wall thickness of the microcapsule can be dynamically adjusted to a preset value by adjusting the input flow rate of the microcapsule outer layer input unit 1.
[0025] See the attached Figure 1 The electrode is arranged inside the output pipe 4, and the lead-out terminal is sent to the control unit. The electrode includes a source electrode 5 and at least one receiving electrode 6, and the source electrode 5 is connected to an AC power supply with an AC power frequency of 1 MHz. During the experiment, the AC charging frequency can be selected from 100 kHz to 10 MHz, or a mixed signal of several different frequencies within the above frequency range.
[0026] In the experiment, two receiving electrodes 6 are arranged symmetrically on both sides of the source electrode 5, with a total of three electrodes. The analysis control unit processes the signals on the two receiving electrodes 6 differentially. One arrangement is that the arrangement direction of the three electrodes is perpendicular to the flow direction of the microcapsules in the output pipe 4 (see Appendix 1), and the two receiving electrodes 6 can be arranged on the same side or both sides of the output pipe 4. Another arrangement is that the arrangement direction of the three electrodes is the same as the flow direction of the microcapsules in the output pipe 4 (see Appendix 1). Figure 2 ), the two receiving electrodes 6 can also be located on the same side or both sides of the output pipe 4. The arrangement direction of the electrodes can also be rotated in the flow direction by an angle, generally 30 to 90 degrees.
[0027] According to the situation that the diameter of the microcapsule is about 100 μm, the electrode gap is also about 100 μm, that is, the distance between the source electrode and the two receiving electrodes is about 100 μm. The diameter of the microcapsule that can be detected is 10 μm-1000 μm, and the corresponding electrode gap is 0.1 to 2 times the diameter of the microcapsule.
[0028] During the preparation of cell culture microcapsules, as the microcapsules flow in the output pipe, the electrodes on the output pipe receive the fluid impedance signal in the output pipe. The analysis control unit obtains the wall thickness of the microcapsules in the current output pipe based on the impedance signal. Since the size parameters of the microcapsules are linearly related to the input flow of the microcapsule outer layer input unit, the input flow can be dynamically adjusted based on the obtained parameters such as the wall thickness, so that the size parameters of the microcapsules meet the predetermined requirements.
[0029] Through experiments, we can obtain microcapsules of different sizes for different input flow rates. The impedance waveforms of multiple microcapsules are shown in the attached figure. Figure 3 ,Each spike waveform represents a microcapsule. According to the positive and negative amplitudes of each spike signal, the parameter electrical diameter D can be obtained. e , electrical diameter D e The value of corresponds to the amplitude of the spike signal. Measure the electrical diameter D e Finally, by establishing a mathematical model, the core size D of the microcapsule is calculated. This can be achieved by the following method: Electrical diameter D e Assume that there is a linear functional relationship between the core size D and D=aD e +b, where a and b are fitting parameters, which need to be determined by fitting experimental data. The electrical diameter D e and core size D are recorded as several categories, each containing multiple data points. To simplify the analysis, the electrical diameter D of each category e The average value of the core large D small is taken as the typical value representing this class. In order to determine the parameters a and b in the model, the least squares method is used for fitting to minimize the error sum between the fitting function and the actual data.e Estimate its core size D.
[0030] Likewise, the calculated dimensions corresponding to the inner diameter and wall thickness can be obtained.
[0031] Through microscopic measurement of the microcapsules for measuring impedance, it was found that the diameter and wall thickness of the microcapsules corresponded one to one with the data calculated from the impedance waveform, and the corresponding relationship could be determined through the data of the two.
[0032] See attached Figure 4 , through a number of different microcapsule electrical diameters and microcapsule core size is linearly related. And the microcapsule core size is directly proportional to the flow rate of gel input.
[0033] The above are only preferred embodiments of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A cell culture microcapsule preparation system, characterized in that: The invention comprises a preparation pipeline, a microcapsule outer layer input unit, a cell input unit and a microcapsule solidified oil input unit arranged at the front end of the preparation pipeline, and is used for preparing microcapsules. The rear end of the preparation pipeline is an output pipeline, and electrodes are arranged in the output pipeline. The electrodes are connected to an analysis control unit, and the analysis control unit obtains parameter information of the microcapsules according to the impedance change in the output pipeline detected by the electrodes. According to the parameter information of the microcapsules, the analysis control unit adjusts the input flow and pressure of the microcapsule outer layer input unit, the cell input unit and the microcapsule solidified oil input unit, and dynamically adjusts the parameter information of the microcapsules to reach a preset value.
2. The cell culture microcapsule preparation system according to claim 1, characterized in that: The electrode comprises a source electrode and at least one receiving electrode, wherein the source electrode is connected to an AC power source, wherein the AC power frequency is 100 kHz to 10 MHz, or a mixed signal of several different frequencies within the above frequency range.
3. The cell culture microcapsule preparation system according to claim 2, characterized in that: There are two receiving electrodes, which are symmetrically arranged on both sides of the source electrode, and the analysis and control unit processes the signals on the two receiving electrodes differentially.
4. The cell culture microcapsule preparation system according to claim 1, characterized in that: The parameter information of the microcapsule includes the external shape, size, wall thickness and number of cells in the microcapsule.
5. The cell culture microcapsule preparation system according to claim 3, characterized in that: The arrangement direction of the electrodes is at an angle to the flow direction of the microcapsules in the output pipe.
6. The cell culture microcapsule preparation system according to any one of claims 1 to 5, characterized in that: The diameter of the microcapsule is 10 μm-1000 μm, and the electrode gap is 0.1 to 2 times the diameter of the microcapsule.
7. The cell culture microcapsule preparation system according to any one of claims 1 to 5, characterized in that: The microcapsule outer layer input by the microcapsule outer layer input unit is sodium alginate gel, the microcapsule solidifying oil input by the microcapsule solidifying oil input unit comprises a mixture of mineral oil, calcium chloride and surfactant, and the sodium alginate is mixed with the calcium chloride in the microcapsule solidifying oil and then solidified to form microcapsules.
8. A method for preparing cell culture microcapsules, using the cell culture microcapsule preparation system according to any one of claims 1 to 7, characterized in that: The steps include: (1) The microcapsule outer layer input unit, the cell input unit and the microcapsule solidified oil input unit generate microcapsules in the preparation pipeline, and the microcapsules flow out through the output pipe of the preparation pipeline; (2) The electrodes on the output pipeline measure the fluid impedance signal in the output pipeline; (3) The analysis control unit obtains parameter information of the microcapsules in the current output pipeline according to the impedance signal; (4) adjusting the input flow and pressure of the microcapsule outer layer input unit, the cell input unit, and the microcapsule solidified oil input unit according to the comparison between the current microcapsule parameter information and the preset value; (5) Repeat steps (2)-(4) to dynamically adjust the parameter information of the microcapsule to reach the preset value.
9. The method for preparing cell culture microcapsules according to claim 8, characterized in that: In the step (3), the parameter information of the microcapsule is the outer diameter and the wall thickness, the impedance signal is a plurality of sharp waves, each sharp wave represents a microcapsule, and the outer diameter and the wall thickness of the microcapsule are calculated according to each sharp wave signal.
10. The method for preparing cell culture microcapsules according to claim 9, characterized in that: In the step (4), the input flow rate and pressure of the microcapsule outer layer input unit are in direct proportion to the outer diameter and wall thickness of the microcapsule. The input flow rate and pressure of the microcapsule outer layer input unit are adjusted according to the comparison between the current outer diameter and wall thickness of the microcapsule and the preset values.