A decellularization method, device and chip based on real-time data acquisition and process control

Abstract

The application provides a kind of based on real-time data acquisition and process regulation decellularization method, device and chip.The device includes: decellularization processing module, for completing decellularization process according to real-time updated configuration parameters, extracting decellularization working fluid sample;Sample detection and data acquisition module is used for real-time completion of DNA and specified decellularization matrix ECM molecule characteristic parameter detection and data acquisition in decellularization working fluid, quantitatively calculates the progress degree of current decellularization process, forms decellularization process information;Data analysis and general control system module is used for analyzing and storing real-time feedback decellularization process information, and controls decellularization process.Experiments show that the method and device of the application can significantly improve the stability and automation degree of decellularization process.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material synthesis technology, and specifically relates to a decellularization method, device and chip based on real-time data acquisition and process control. Background Technology

[0002] The extracellular matrix (ECM) is a crucial component of human and animal tissues and organs, containing a rich variety of biomolecules such as collagen, adhesion glycoproteins, and proteoglycans. The ECM forms a network-like structure outside the cell, maintaining close contact with the cell and connecting to the cytoskeleton and even the nucleus through cell surface receptors. It provides information in the microcirculation and plays a vital role in cell growth, differentiation, adhesion, and movement. Furthermore, the ECM exhibits significant differences in biochemical, physical, and mechanical characteristics among different tissues and organs. This specificity plays a crucial role in regulating cell behavior and fate, as well as tissue and organ function, and is decisive in tissue development, homeostasis, and disease progression.

[0003] Decellularized extracellular matrix (dECM) is a biomaterial derived from natural tissues by removing cellular components while preserving the structure, composition, and mechanical properties of the tissue / organ's ECM, such as the microscopic arrangement of structural proteins, diverse functional proteins, growth factors, and soluble factors. Compared to traditional bioactive materials, tissue-specific dECM can mimic the composition and structure of tissue / organ ECM to the greatest extent possible, offering significant advantages in regulating cellular responses and tissue remodeling. Currently, dECM is widely used in scientific research and clinical implantation and is considered one of the most promising biomedical materials in terms of application potential and market prospects.

[0004] To fully leverage the tissue specificity and bioactivity advantages of natural organ-derived cellular membranes (ECMs) and avoid triggering immune rejection after in vivo, ideal dECMs should retain the essential components of ECM to the greatest extent possible while reducing the content of immunogenic substances (such as DNA and α-Gal) to below risk standards. Current mainstream decellularization processes include physical treatments (such as repeated freeze-thaw cycles and mechanical vibration), chemical treatments (acids, alkalis, ions, chelates, etc.), enzymatic treatments (trypsin, nucleases, etc.), and processes combining multiple methods. The main purpose is to lyse and release the cellular contents and remove them from the organ-derived tissue to obtain dECM materials.

[0005] Currently, the decellularization of human and animal tissues and organs typically relies on experience and accumulated techniques, resulting in standardized processes. Furthermore, the content of key components in dECM materials can only be determined through quantitative characterization of the final decellularized product. Real-time monitoring and data collection of component content and other information during the decellularization process are not possible, and there is a lack of intelligent, automated, integrated dECM material preparation systems capable of flexibly controlling the decellularization process. This is specifically reflected in the following aspects:

[0006] (1) The decellularization process of human and animal tissues and organs typically involves multiple steps (such as impurity removal, oil removal, decellularization, cleaning, and sterilization), which is complex and time-consuming (several days to several weeks). The process details of each step (processing time, processing intensity, etc.) directly affect the quality of dECM material products. Currently, the evaluation of dECM material products generally adopts the pharmaceutical industry recommended standards (such as YY / T0606.25-2014, YY / T 1561-2017, YY / T 1453-2016, etc.) to evaluate the residual DNA, α-Gal, and type I collagen content in the final dECM material product, and iteratively optimizes the decellularization process based on the evaluation results, thereby improving the quality of dECM materials. However, this method of optimization based on the test results of the end product is difficult to accurately control every step of the decellularization process and has low optimization efficiency. At the same time, given that the properties of raw materials (natural tissues and organs) from different sources and batches are different, even a decellularization process that has been optimized and determined multiple times cannot guarantee the product stability and product quality of dECM materials.

[0007] (2) According to literature, North Carolina State University in the United States used the absorbance of DNA molecules in the 260 nm wavelength range and a UV spectrophotometer to determine the concentration of DNA molecules in the decellularization working solution, thereby initially optimizing the decellularization process by plotting DNA release curves. However, since the chemical solvents and protein molecules in the solution also have strong absorbance in the 230-280 nm wavelength range, there is serious signal interference, which makes the method inaccurate in monitoring DNA concentration. It can only give a vague trend curve and cannot accurately calculate the actual DNA content. Therefore, it is difficult to accurately judge the decellularization process and make flexible adjustments.

[0008] (3) Most existing decellularization processes require quantitative analysis of various monitoring indicators after the final dECM material product is produced. This requires considerable manpower and resources to complete sampling and testing, resulting in some losses in production costs and output efficiency. If the decellularization process can be controlled and optimized in real time through sample testing, data collection and analysis feedback during the decellularization process steps, the production efficiency of dECM materials can be greatly improved and the cost reduced.

[0009] It can be seen that the existing technology has the following three main problems:

[0010] (1) The feedback data was collected using ultraviolet spectrophotometry, whose accuracy is highly dependent on the purity of the DNA in the solution. The decellularization working solution contains a large amount of impurities such as organic solvents, dissociative salts, and proteins, which severely affect the absorbance measurement results of DNA molecules, leading to significant errors. The feedback data can only be used for qualitative analysis and cannot be used as a quantitative reference. Therefore, this method cannot ensure that each batch of dECM material has the same or similar component content, cannot guarantee product stability, and cannot be used to improve the efficiency of the decellularization process.

[0011] (2) The overlap of the absorbance bands of impurities and DNA molecules leads to errors in the determination of the residual amount of harmful substances such as organic solvents and liquid salts, making it impossible to use as a reference for evaluating the safety of dECM materials. At the same time, the overlap of the absorbance bands of protein molecules and DNA makes it impossible to distinguish the residual amount of DNA and important ECM macromolecular components. Therefore, this method is not helpful for the clear quantitative evaluation standard of DNA residual amount below 50 ng / mg in relevant industry standards, and it is difficult to improve the safety of dECM materials.

[0012] (3) The qualitative rather than quantitative information feedback model cannot collect accurate datasets, making it difficult to establish mathematical models and intelligent decision-making mechanisms. Therefore, this method cannot be used to develop intelligent and precise decellularization processes.

[0013] In summary, existing methods for preparing dECM materials cannot control each step of the decellularization process in real time, and generally adopt standardized decellularization processes, which cannot achieve efficient, precise, and customized production of dECM materials. Summary of the Invention

[0014] In view of this, the purpose of this invention is to propose a decellularization method, device and chip based on real-time data acquisition and process control, so as to improve the efficiency of decellularization processing.

[0015] This invention aims to address the challenge of real-time monitoring of the decellularization process of human and animal-derived tissues and organs. It proposes a decellularization method based on real-time data acquisition and analysis, making the blind spot between upstream raw materials (natural tissues and organs) and downstream products (dECM materials) transparent. Furthermore, it establishes an intelligent decision-making model through machine learning algorithms, enabling flexible control over the process during decellularization. Simultaneously, this invention designs a fully automated decellularization device system capable of automatically supplying, discharging, and extracting the decellularization working solution according to a computer-controlled program. A microfluidic chip-based detection device quantifies and collects data from the extracted trace amounts of decellularization working solution samples, and an intelligent decision-making model is established based on the dataset. This achieves efficient, precise, and intelligent decellularization, ultimately leading to the stable mass production of high-quality dECM materials.

[0016] Specifically, the technical problems to be solved by this invention cover the following aspects:

[0017] a) The characteristics of human and animal-derived tissues and organs (e.g., size, thickness, protein content, activity, etc.) are closely related to the donor's age, sex, physiological and disease status, exhibiting variability that is difficult to control. Currently, the decellularization methods used in the industry mostly employ standardized processes derived from accumulated experience. When dealing with tissues and organs with significant differences, batch-to-batch instability is unavoidable. Therefore, stabilizing and improving the quality of dECM materials helps to fully realize their bioactivity and biological functions.

[0018] (b) Existing dECM material products on the market, such as dermal, fat, and small intestine dECM, typically use a DNA residue level of less than 50 ng / mg as the testing standard. Due to the lack of perfect industry standards, a common practice to achieve this goal is to increase the intensity and duration of decellularization. However, excessive decellularization not only reduces dECM production efficiency but also leads to the loss of key ECM components (such as collagen and glycosaminoglycans), severely affecting the quality of dECM materials. Therefore, intelligent and precise decellularization methods that monitor and provide feedback on the timeliness of the decellularization process in real time can improve the safety and efficiency of dECM products.

[0019] c) Existing decellularization methods (physical, chemical, and enzymatic treatments) have varying decellularization effects. How to fully utilize the advantages of these methods and optimize the decellularization process for different tissues and organs is a pressing issue. Therefore, this study collects real-time data on the decellularization efficiencies of the aforementioned methods and uses machine learning algorithms to construct optimization models. This enables intelligent formulation of decellularization process routes. Furthermore, an intelligent, automated, integrated dECM material preparation system is designed and developed to precisely control the entire process from raw materials to dECM material products, thereby achieving intelligent, precise, and customized production of dECM materials.

[0020] To achieve the above objectives, this invention proposes a decellularization method based on real-time data acquisition and process control, comprising:

[0021] Obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be processed to form a basic reference dataset;

[0022] Start the decellularization process according to the specified configuration parameters;

[0023] At the preset decellularization treatment time points, samples were taken from the decellularization working solution to collect and measure the DNA and specified decellularization matrix ECM molecule content data.

[0024] The DNA and specified decellularized matrix ECM molecule content data are fitted in real time to obtain the real-time fitting result, which is then compared with the basic reference dataset to obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution.

[0025] Based on the real-time fitting results and / or the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution, the current progress of the decellularization process is quantitatively calculated to obtain decellularization process information.

[0026] Feedback is provided based on the decellularization process information, and the specified configuration parameters during the decellularization process are adjusted in real time.

[0027] The decellularization process ends when the progress stabilizes or a specified target is reached.

[0028] In some embodiments, the characteristic parameters include quality parameters.

[0029] In some embodiments, characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated are obtained to form a basic reference dataset, including:

[0030] The concentrations of DNA and specified decellularized matrix ECM molecules in the tissue to be treated, as well as the mass change rate of the lyophilized tissue to be treated, were determined.

[0031] Based on the mass change rate, the actual mass of DNA and specified decellularized matrix ECM molecules contained in the tissue to be treated is calculated to form the basic reference dataset.

[0032] In some embodiments, at preset decellularization treatment time points, samples are taken from the decellularization working solution to collect and measure the content data of DNA and specified decellularized matrix ECM molecules; the DNA and specified decellularized matrix ECM molecule content data are fitted in real time to obtain real-time fitting results, which are compared with the basic reference dataset to obtain characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularization working solution, including:

[0033] At preset time intervals, the decellularized working solution is sampled to obtain working samples. The working samples are then mixed with reactants to label DNA molecules and / or the specified decellularized matrix ECM molecules, resulting in a labeled mixture.

[0034] Based on the aforementioned basic reference dataset, the actual concentration of DNA and specified decellularized matrix ECM molecules in the decellularized working solution is obtained through the characteristic parameters of the labeled mixture.

[0035] The actual mass of DNA and specified decellularized matrix ECM molecules contained in the decellularized working solution is calculated based on the actual concentration and volume of the decellularized working solution, thus forming characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution.

[0036] In some embodiments, based on the real-time fitting results and / or the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularization working solution, the current progress of the decellularization process is quantitatively calculated to obtain decellularization process information, including:

[0037] At preset time intervals, the actual mass of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution is fitted.

[0038] In the fitting process, through formula M T / M P *100% quantitative calculation of the current progress of the decellularization process, where M T M represents the actual mass of DNA or a specified decellularized matrix ECM molecule in the decellularized working solution. PThe actual mass of DNA or specified decellularized matrix ECM molecules in the tissue to be treated;

[0039] By using interpolation, the actual mass of DNA and specified decellularization matrix (ECM) molecules in the tissue to be treated and the decellularization working solution are compared to obtain decellularization process information.

[0040] In some embodiments, the configuration parameters during the decellularization process are adjusted in real time based on feedback from the decellularization process information, including:

[0041] Based on the decellularization process information, determine the effectiveness of the decellularization process and the residual amount of the specified reagent;

[0042] The actual mass of DNA and specified decellularized matrix ECM molecules is input into the decellularized working solution. A decision algorithm is constructed based on the progress level using a machine learning algorithm, and the configuration parameters are trained to obtain the configuration parameters.

[0043] Adjust the decellularization process according to the configuration parameters.

[0044] To achieve the above objectives, this invention proposes a decellularization device based on real-time data acquisition and process control, comprising:

[0045] The decellularization module is used to complete the decellularization process and extract the decellularized working solution sample according to the real-time updated configuration parameters.

[0046] The sample detection and data acquisition module is used to complete the detection and data acquisition of characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularization working solution in real time, quantify the progress of the current decellularization process, and form decellularization process information.

[0047] The data analysis and overall control system module is used to analyze and store the real-time feedback information on the decellularization process and to control the decellularization process.

[0048] In some embodiments, the decellularization module includes:

[0049] A decellularized working solution classification and storage device, consisting of at least one independent storage tank, is used to store different types of decellularized working solutions;

[0050] Liquid delivery unit, each of the liquid delivery units is connected to a corresponding liquid storage tank for delivering decellularized working solution;

[0051] A decellularization processing container, connected to the liquid delivery unit, is used to receive the decellularization working solution delivered by the liquid delivery unit and to perform decellularization processing.

[0052] A control unit, connected to the liquid delivery unit, is used to control the liquid delivery unit to deliver the decellularization working solution to the decellularization processing container according to the decellularization configuration parameters; a decellularization controller is used to regulate the decellularization process according to the specified decellularization processing configuration parameters.

[0053] In some embodiments, the decellularization processing module further includes:

[0054] Waste liquid collection unit, used to collect the waste liquid generated during the decellularization process.

[0055] In some embodiments, the decellularization processing module further includes: a liquid extraction unit, configured to extract a specified volume of decellularization working solution from the outlet of the decellularization processing container according to specified configuration parameters.

[0056] In some embodiments, the decellularization processing module further includes:

[0057] A filter is used to filter the decellularized working solution extracted by the extraction unit.

[0058] In some embodiments, the liquid delivery unit includes a liquid delivery pump, and the liquid extraction unit includes a liquid extraction pump.

[0059] In some embodiments, the sample detection and data acquisition module includes:

[0060] The calibration and detection unit includes at least one microfluidic chip for mixing, detecting, and cleaning the decellularized working solution, and transmitting the detection results to the data analysis and overall control system module in real time.

[0061] In some embodiments, the data analysis and overall control system module includes:

[0062] The data storage unit is used to store the sample data collected in real time by the sample detection and data acquisition module, and to establish or update the decellularization process information.

[0063] The control and computing unit is used to solve and / or fit, and to establish a decision model based on machine learning algorithms, calculate configuration parameters, and feed back the configuration parameters to the decellularization processing module.

[0064] This invention also proposes a microfluidic chip for decellularization, characterized in that the microfluidic chip is made of a low / non-adsorbent material, comprising:

[0065] A working fluid inlet is provided to allow the decellularized working fluid to enter the chip.

[0066] The detection solution inlet is used to receive the decellularized detection solution into the chip;

[0067] A mixing unit, connected to the working solution inlet and the detection solution inlet, is used to mix the decellularized working solution and the decellularized detection solution;

[0068] The sample reading unit, connected to the mixing unit, is used to read sample information.

[0069] In some embodiments, the microfluidic chip for decellularization further includes:

[0070] The waste liquid pool is connected to the sample reading unit and is used to discharge waste liquid after reading the sample information.

[0071] In some embodiments, the microfluidic chip for decellularization further includes:

[0072] The cleaning fluid inlet is connected to the mixing channel and is used for the cleaning fluid to enter the chip.

[0073] In summary, the beneficial effects of this invention are: (1) It breaks through the limitations of real-time quantitative and accurate detection of dECM material components, thereby improving product stability and production efficiency. (2) It can distinguish the effects of DNA and organic solvents, dissociative salts, enzymes, and other decellularization working solution components on fluorescence intensity. Therefore, this method can separately determine the residual amounts of DNA and toxic substances in dECM materials, thereby improving the safety of dECM materials by optimizing the decellularization process. (3) It utilizes the method designed in this invention for automated decellularization processing. Attached Figure Description

[0074] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in the invention and should not be construed as limiting the scope of the invention.

[0075] Figure 1 A flowchart illustrating a decellularization method based on real-time data acquisition and process control according to an embodiment of the present invention is shown.

[0076] Figure 2 A schematic diagram of a decellularization method based on real-time data acquisition and process control according to an embodiment of the present invention is shown.

[0077] Figure 3 A flowchart illustrating the decellularization process of porcine dermis according to an embodiment of the present invention is shown.

[0078] Figure 4 A schematic diagram of a decellularization system based on real-time data acquisition and process control according to an embodiment of the present invention is shown.

[0079] Figure 5A schematic diagram of a decellularization system based on real-time data acquisition and process control according to another embodiment of the present invention is shown.

[0080] Figure 6 A microfluidic chip structure for decellularization processing according to an embodiment of the present invention is shown.

[0081] Figure 7A A comparison chart of theoretical calculation and actual measurement errors according to an embodiment of the present invention is shown.

[0082] Figure 7B Another comparison chart showing the theoretical calculation and actual measurement error according to an embodiment of the present invention is shown.

[0083] Figure 8A The image shows the decellularization effect of the traditional process.

[0084] Figure 8B The diagram shows the decellularization effect according to an embodiment of the present invention.

[0085] Figure 9 The diagram illustrates the effect of removing immunogenic substances according to an embodiment of the present invention.

[0086] Figure 10 A graph showing the toxicity test results according to an embodiment of the present invention is provided. Detailed Implementation

[0087] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0088] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0089] In the implementation of the technical solution of this invention, the contents of DNA and important ECM macromolecular components in the tissues and organs to be treated are first measured and calculated. Then, a rapid quantitative detection method with extremely low susceptibility to impurities is used to accurately measure the concentration of DNA and important ECM macromolecular components in the working solution during the decellularization process. Then, the contents of residual DNA and important ECM macromolecular components in the dECM material are indirectly calculated and solved by the difference method. Thus, the content data of each component of the dECM material are collected in real time. Subsequently, an accurate decellularization process curve is generated by data fitting. And an intelligent decision-making algorithm can be constructed based on machine learning using the collected data, thereby realizing real-time control of the decellularization process and targeted and evidence-based process control to obtain high-quality and high-efficiency dECM products.

[0090] Figure 1 A flowchart illustrating a decellularization method based on real-time data acquisition and process control according to an embodiment of the present invention is shown. Figure 1 As shown, this decellularization method based on real-time data acquisition and process control includes:

[0091] Step S11: Obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated to form a basic reference dataset.

[0092] In one implementation, the characteristic parameters include quality parameters.

[0093] Specifically, the characteristic parameters can be quality parameters, or any other parameters that can measure the decellularization process, such as volume, specific gravity, or quantity.

[0094] The specified decellularized matrix ECM molecule can be an ECM macromolecule target such as glycosaminoglycans.

[0095] In one implementation, characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated are obtained to form a basic reference dataset, including:

[0096] The concentrations of DNA and specified decellularized matrix ECM molecules in the tissue to be treated, as well as the mass change rate of the lyophilized tissue to be treated, were determined.

[0097] Based on the mass change rate, the actual mass of DNA and specified decellularized matrix ECM molecules contained in the tissue to be treated is calculated to form the basic reference dataset.

[0098] Specifically, the base reference dataset is used to compare the progress of the current decellularization process and can include two types of data. One type is the latest sample data obtained through the current processing, as well as derived data such as fitted curves obtained from these data. The other type is existing technical documents, public publications, or records of empirical values, such as records in textbooks, sample datasets, patent documents, and other literature, as well as datasets derived from these data.

[0099] Step S12: Start the decellularization process according to the specified configuration parameters.

[0100] Specifically, configuration parameters refer to any parameters that can adjust the decellularization process, such as temperature and concentration. Since the key aspect of this invention lies in real-time adjustment of the decellularization process, these configuration parameters can be dynamically adjusted in real time upon receiving an adjustment command, thereby improving the efficiency of the decellularization process.

[0101] Step S13: At the preset decellularization treatment time point, take samples from the decellularization working solution, collect and measure the DNA and specified decellularization matrix ECM molecule content data.

[0102] Step S14: Perform real-time fitting on the DNA and specified decellularized matrix ECM molecule content data to obtain the real-time fitting result, compare it with the basic reference dataset, and obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution.

[0103] In one implementation, steps S13 and S14 include:

[0104] At preset time intervals, the decellularized working solution is sampled to obtain working samples. The working samples are then mixed with reactants to label DNA molecules and / or the specified decellularized matrix ECM molecules, resulting in a labeled mixture.

[0105] Based on the aforementioned basic reference dataset, the actual concentration of DNA and specified decellularized matrix ECM molecules in the decellularized working solution is obtained through the characteristic parameters of the labeled mixture.

[0106] The actual mass of DNA and specified decellularized matrix ECM molecules contained in the decellularized working solution is calculated based on the actual concentration and volume of the decellularized working solution, thus forming characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution.

[0107] Specifically, the reactants may include staining agents capable of recognizing and labeling DNA molecules and / or specific decellularized matrix ECM molecules, such as staining agents like Hoechst.

[0108] Step S15: Based on the real-time fitting results and / or the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution, quantify the progress of the current decellularization process to obtain decellularization process information.

[0109] In one implementation, step S15 includes:

[0110] At preset time intervals, the actual mass of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution is fitted.

[0111] In the fitting process, through formula M T / M P *100% quantitative calculation of the current progress of the decellularization process, where M TM represents the actual mass of DNA or a specified decellularized matrix ECM molecule in the decellularized working solution. P The actual mass of DNA or specified decellularized matrix ECM molecules in the tissue to be treated;

[0112] By using interpolation, the actual mass of DNA and specified decellularization matrix (ECM) molecules in the tissue to be treated and the decellularization working solution are compared to obtain decellularization process information.

[0113] Specifically, the current progress of the decellularization process can be measured using formula M. T / M P *100% quantitative calculations are performed. The decellularization process can be measured by calculating changes in DNA in the decellularization working solution, or by calculating changes in specific decellularization matrix ECM molecules in the decellularization working solution. Alternatively, a combination of changes in both DNA and specific decellularization matrix ECM molecules can be used to measure the decellularization process. In other words, when M... T When M represents the actual mass of DNA in the decellularized working solution P It should be the actual mass of DNA in the tissue to be processed; when M T When specifying the actual mass of decellularized matrix ECM molecules in the decellularized working solution, M P The actual mass of the decellularized matrix ECM molecules in the tissue to be treated should be specified. When considering changes in both DNA and specified decellularized matrix ECM molecules in the decellularization working solution to measure the decellularization process, the changes in DNA and specified decellularized matrix ECM molecules in the decellularization working solution can be calculated separately first, and then normalized using methods such as weighting.

[0114] Step S16: Feedback is provided based on the decellularization process information, and the specified configuration parameters in the decellularization process are adjusted in real time.

[0115] Specifically, based on the feedback of the decellularization process information, the current decellularization effect can be determined, and then one or more of the configuration parameters such as the type and concentration of the decellularization solution, the processing time, and the ambient temperature and humidity can be calculated. These configuration parameters are then transmitted to the control unit to achieve dynamic control and adjustment of the decellularization process, thereby improving the efficiency of the decellularization process.

[0116] In one implementation, step S16 includes:

[0117] Based on the decellularization process information, determine the effectiveness of the decellularization process and the residual amount of the specified reagent;

[0118] The actual mass of DNA and specified decellularized matrix ECM molecules is input into the decellularized working solution. A decision algorithm is constructed based on the progress level using a machine learning algorithm, and the configuration parameters are trained to obtain the configuration parameters.

[0119] Adjust the decellularization process according to the configuration parameters.

[0120] Specifically, the designated reagents may include decellularization working solutions such as disinfectants, DNase reagents, Triton X-100 reagents, and enzymes.

[0121] Step S17: When the progress stabilizes or the specified target is reached, the decellularization process is terminated.

[0122] Specifically, "reaching a stable state" means that the entire decellularization process reaches a stable state over time without significant progress. "Achieving a specified target" means that the decellularization process reaches a pre-defined level, for example, a specified DNA concentration below 50 ng / mg.

[0123] In one implementation, the above steps can be specifically carried out as follows:

[0124] 1) Determine the actual mass of DNA and important ECM macromolecules in the tissue to be treated. Using fluorescence staining, ELISA (Enzyme-Linked Immunosorbent Assay), and characterization methods outlined in industry standards (YY / T 0606.25-2014, YY / T 1561-2017, YY / T 1453-2016 for tissue engineering medical products), accurately determine the concentration of DNA and important ECM macromolecules (glycosaminoglycans, collagen, elastin, etc.) in natural tissues and organs, as well as the mass change rate (L) after lyophilization. Based on this, calculate the mass (M) of the tissue to be treated. O The actual mass (M) of the DNA and important ECM macromolecular components contained in it. P ).

[0125] 2) Real-time measurement and calculation of the content of DNA and important ECM macromolecules in the decellularization working solution. At preset decellularization time points (e.g., every 15 minutes), a micro-sample (10 μL) of the decellularization working solution is taken and mixed with dyes to label target substances such as DNA molecules and ECM macromolecules. The fluorescence and absorbance values ​​of the mixture are recorded and compared with the data curve of standard samples to accurately obtain the actual concentration of DNA and important ECM macromolecules in the current decellularization working solution (C0). PThen, combining this with the total volume of the decellularized working solution, the actual mass (M) of DNA and important ECM macromolecules contained in the current decellularized working solution is calculated. T ).

[0126] 3) Real-time feedback data fitting and component content determination in dECM. Data obtained from sampling at continuous time points will be fitted and solved, and curves will be plotted. Wherein, M... T / M P *100% represents the real-time decellularization process P. Simultaneously, M is compared using the difference method. P With M T The content of DNA and important ECM macromolecules in the current dECM material can be calculated using the following formula (C). P P and C are obtained by sampling based on the duration node. P The values ​​are then subjected to curve fitting.

[0127]

[0128] 4) Intelligent process setting and implementation based on real-time decellularization process. By referring to real-time feedback data and curve trends of DNA and important ECM macromolecule content in dECM materials, the timeliness of the current decellularization process and the residual amount of toxic reagents can be determined. Simultaneously, an intelligent decision-making algorithm is constructed from the feedback dataset using machine learning algorithms to intelligently control and optimize the decellularization process. This not only improves the safety of dECM material products but also enables the preparation of customized dECM materials with specific DNA and important ECM macromolecule contents for different application scenarios and needs. Furthermore, because this method allows for flexible control of the decellularization process based on real-time data of relevant component content in dECM materials, it has universal applicability to the development of dECM materials from different sources and types of tissues and organs, eliminating the need for process accumulation and significantly improving development efficiency and success rate.

[0129] Figure 2 A schematic diagram of a decellularization method based on real-time data acquisition and process control according to an embodiment of the present invention is shown. Figure 2 Taking porcine dermal dECM material as an example, the technical solution processing procedure of this invention is as follows:

[0130] 1) If 100 mg of porcine dermal tissue is taken, and its freeze-dried mass is determined to be 60 mg, then its freeze-drying mass change rate L = 60%. If the DNA content is determined to be 500 ng / mg and the glycosaminoglycan content is 100 μg / mg, then the freeze-dried mass of 1000 mg of the treated natural porcine dermal tissue is 600 mg, and the total mass of DNA and glycosaminoglycan is M. P (DNA)=1000*60%*500=300ug, MP (GAGs)=1000*60%*100=60mg;

[0131] 2) Assuming that the DNA concentration detected in 1000 mL of Triton X-100 decellularization working solution is 50 ng / ml and 100 ng / ml at 15 minutes and 30 minutes after the start of decellularization, respectively, then the total mass of DNA in the solution is M. T The DNA content in the dermal dECM material was 250 μg and 200 μg, respectively, with 50 μg and 100 μg of DNA respectively. The DNA concentration C... P The glycosaminoglycan (DNA) concentrations were 416.47 ng / mg and 333 ng / mg, respectively, and the decellularization progress percentages (P, DNA) were 16.67% and 33.33%, respectively. Similarly, assuming that the glycosaminoglycan concentrations detected in 1000 mL of Triton X-100 decellularization working solution were 1 μg / mL and 3 μg / mL, respectively, 15 and 30 minutes after the start of the decellularization process, then the total mass of glycosaminoglycans (M) would be... T (GAGs) were 1 mg and 3 mg respectively, and the glycosaminoglycan content in the dermal dECM material was 59 mg and 57 mg respectively, with glycosaminoglycan concentration C. P The concentrations of the two glycoproteins (GAGs) were 98.3 μg / mg and 95 μg / mg, respectively, with loss rates of 1.7% and 5%.

[0132] 3) During the decellularization process, an automated device is used to sample and test the decellularization working solution every 5 minutes. By calculating and solving the test data and fitting the decellularization process curve, real-time monitoring of DNA and important ECM macromolecular components in the dECM material can be achieved. The sampling time can be flexibly set according to different needs, with a minimum time interval of 1 minute.

[0133] 4) Based on the changing patterns of the decellularization process curve, the timeliness of the current decellularization process can be determined. For example, if the reduction rate of DNA content in the dECM material is less than 5% for 15 consecutive minutes, it can be determined that the current decellularization process has reached saturation. Continuing to extend the processing time of the current process will not only make it difficult to achieve the purpose of decellularization, but may also cause loss to other important ECM macromolecular components in the material. Therefore, it is advisable to switch to the next decellularization process. In addition to determining the effectiveness of the current decellularization process step, the decellularization process curve can also be used to determine the residual amount of toxic biochemical reagents and whether the immune risk substances in the dECM material have been reduced to below the risk standard (e.g., DNA content below 50 ng / mg), which can effectively improve the safety of the dECM material. At the same time, the decellularization process curve can also provide real-time feedback on the content of other important ECM macromolecular components contained in the dECM material. This allows for the integration of machine learning to develop intelligent decision-making algorithms for decellularization processes tailored to different scenario requirements (e.g., high collagen content, high glycosaminoglycan content, etc.), thereby achieving customized production of dECM materials.

[0134] Figure 3 A flowchart illustrating the decellularization process of porcine dermis according to an embodiment of the present invention is shown. Figure 3 As shown, taking porcine dermal decellularization as an example, the specific process of the intelligent and precise decellularization technology solution designed in this invention is as follows:

[0135] Step 1: Pretreatment of pig skin tissue. Fresh pig skin tissue is cut into 5cm x 5cm pieces. Epidermal and adipose tissue are removed, retaining the dermis. 100ml of Trypsin (1%–2.5%) and 2ml of 0.5M ethylenediaminetetraacetic acid (EDTA) (0.1M–1M) are added to an aqueous solution to obtain a Trypsin and EDTA aqueous solution. The solution is magnetically stirred for 2–6 hours (100–400 rpm / min) to thoroughly remove dust, blood, and residual epidermal and adipose cells. After 2–6 hours, a portion is freeze-dried, and the rate of change in mass (L) after freeze-drying is calculated. Another portion is further decellularized.

[0136] Step 2: Distilled Water (DW) Washing. Add 1000 mL of pure water to a beaker, place the tissue in it, and wash with a magnetic stirrer to remove any remaining Trypsin and EDTA from Step 1. Sample the DW decellularized working solution every 15 minutes. After sampling, calibrate the samples using the Hoechst fluorescence method and compare the results with the DW calibration values. If the two values ​​are similar, it indicates that there are no residual enzymes or chemical reagents in the dECM material, and Step 2 can be stopped.

[0137] Step 3: Triton-X100 Treatment. Add 100 ml of 10x phosphate-buffered saline (PBS), 10–50 ml of Triton-X100 solution, and 50–200 ml of 0.5 M EDTA solution to a beaker. Then add 650–840 ml of pure water and mix thoroughly to obtain the Triton X-100 decellularized working solution. Add the dECM material treated in Step 2 to this solution and wash with a magnetic stirrer. Sample the decellularized working solution every 15 minutes. After sampling, calibrate the samples using fluorescence spectrometry and compare the results with the calibration values ​​of the Triton X-100 decellularized working solution. Calculate the total DNA mass (M) in the Triton X-100 decellularized working solution. T (DNA) and DNA concentration C P (DNA) Based on data fitting, a decellularization process curve is plotted until the curve reaches a plateau, indicating that the decellularization efficiency of the current Triton X-100 decellularization working solution is close to saturation, and step three can be stopped. Simultaneously, the DNA content in the dECM material is determined using the difference method. If it exceeds the risk standard (50 ng / mg), step three can be repeated.

[0138] Step 4: Rinse with Distilled Water (DW). Add 1000 mL of pure water to a beaker, place the tissue in it, and wash with a magnetic stirrer to remove any residual Triton X-100 reagent from Step 1. Sample the DW decellularized working solution every 15 minutes. After sampling, calibrate the samples using the Heochst fluorescence method and compare the results with the DW calibration values. If the two values ​​are similar, it indicates that there is no residual Triton X-100 reagent in the dECM material, and Step 4 can be stopped.

[0139] Step 5: Nuclease (DNase) treatment. Add 100ml of 10x concentration PBS solution, 10ml of penicillin / streptomycin solution, and 890ml of pure water to a beaker. Finally, add 5-50KU of DNase and mix thoroughly to prepare the DNase decellularization working solution. Add the dECM material treated in Step 2 to this solution and wash with a magnetic stirrer. Sample the DNase decellularization working solution every 15 minutes. After sampling, calibrate the samples using fluorescence spectrometry and compare the results with the calibration value of the original DNase decellularization working solution as a control. Calculate the total DNA mass M in the DNase decellularization working solution. T (DNA) and DNA concentration C P(DNA) Based on data fitting, plot the decellularization process curve until the curve reaches a plateau, indicating that the current DNase decellularization working solution has nearly reached saturation, and step five can be stopped. Simultaneously, use the difference method to determine the DNA content in the dECM material. If it exceeds the risk standard (50 ng / mg), step five can be repeated.

[0140] Step Six: PBS Washing. Add 100 ml of 10x concentrated PBS solution, 10 ml of penicillin / streptomycin solution, and 890 ml of pure water to a beaker. Place the tissue in the solution and wash with a magnetic stirrer to remove any residual DNase from Step Five. Sample the PBS decellularization working solution periodically. After sampling, perform Hoechst fluorescence assay, using the fluorescence intensity of PBS as a control. If the two are similar, it indicates that there is no residual DNase in the dECM material, and Step Six can be stopped.

[0141] Step 7: Sterilization and Disinfection. Add 100ml of 10x concentration PBS solution and 750–840ml of pure water to a beaker. Add 10–50ml of 15% peracetic acid solution and 50–100ml of 75% alcohol, mix thoroughly to prepare the sterilization and decellularization working solution. Add the tissue to this solution and wash for 2–6 hours to kill any residual bacteria, viruses, and other microorganisms in the dECM material. Sample the sterilization and decellularization working solution periodically. After sampling, use the Hoechst fluorescence method for calibration, using the fluorescence intensity of the original sterilization and decellularization working solution as a control. Calculate the total DNA mass M in the sterilization and decellularization working solution. T (DNA) and DNA concentration C P (DNA) The DNA content in the dECM material was determined by the difference method, and the decellularization process curve was plotted based on the data fitting.

[0142] Step 8: PBS Treatment. Add 100ml of 10x concentrated PBS, 10ml of penicillin / streptomycin solution, and 890ml of pure water to a beaker. Place the tissue in the solution and wash with a magnetic stirrer to remove any residual peracetic acid and alcohol from Step 7. Sample the PBS decellularization working solution periodically. After sampling, use the Hoechst fluorescence method for calibration, using the fluorescence intensity of the PBS as a control. If the two are similar, it indicates that there are no residual disinfectant reagents in the dECM material, and Step 8 can be stopped. This completes the entire decellularization process.

[0143] Figure 4 A schematic diagram of a decellularization device based on real-time data acquisition and process control according to an embodiment of the present invention is shown. Figure 4 As shown, this decellularization system based on real-time data acquisition and process control can be divided into the following components:

[0144] The decellularization module 41 is used to complete the decellularization process and extract the decellularization working solution sample according to the real-time updated configuration parameters.

[0145] The sample detection and data acquisition module 42 is used to complete the detection and data acquisition of characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularization working solution in real time, quantify the progress of the current decellularization process, and form decellularization process information.

[0146] The data analysis and overall control system module 43 is used to analyze and store the real-time feedback information on the decellularization process and to control the decellularization process.

[0147] In one embodiment, the decellularization processing module 41 includes:

[0148] The decellularized working solution classification and storage device 411 consists of at least one independent storage tank for storing different types of decellularized working solutions;

[0149] Liquid delivery unit 412, each of the liquid delivery units is connected to a corresponding liquid storage tank, for delivering decellularized working solution;

[0150] The decellularization processing container 413 is connected to the liquid delivery unit and is used to receive the decellularization working solution delivered by the liquid delivery unit and perform decellularization processing.

[0151] Control unit 414, connected to the liquid delivery unit, is used to control the liquid delivery unit to deliver the decellularization working solution to the decellularization processing container according to the decellularization configuration parameters;

[0152] The decellularization controller 415 is used to regulate the decellularization process according to specified decellularization processing configuration parameters.

[0153] In one embodiment, the decellularization processing module 41 further includes:

[0154] Waste liquid collection unit 416 is used to collect the waste liquid generated during the decellularization process.

[0155] In one embodiment, the decellularization processing module 41 further includes:

[0156] The liquid extraction unit 417 is used to extract a specified volume of decellularized working solution from the outlet of the decellularization processing container according to specified configuration parameters.

[0157] In one embodiment, the decellularization processing module 41 further includes:

[0158] Filter 418 is used to filter the decellularized working solution extracted by the extraction unit.

[0159] In one embodiment, the liquid delivery unit 412 includes a liquid delivery pump.

[0160] Of course, the liquid delivery unit 412 can also be other devices with liquid delivery functions besides the liquid delivery pump, or a combination of devices.

[0161] In one embodiment, the pumping unit 417 includes a pumping pump.

[0162] Of course, the liquid pumping unit 417 can also be other devices with liquid pumping functions besides the liquid pump, or a combination of devices.

[0163] In one embodiment, the sample detection and data acquisition module 42 includes:

[0164] The calibration and detection unit 421 includes at least one microfluidic chip for mixing, detecting, and cleaning the decellularized working solution, and transmitting the detection results to the data analysis and overall control system module in real time.

[0165] In one implementation, the data analysis and overall control system module 43 includes:

[0166] Data storage unit 431 is used to store sample data collected in real time by the sample detection and data acquisition module, and to establish or update the decellularization process information;

[0167] The control calculation unit 432 is used to solve and / or fit, and to establish a decision model based on the machine learning algorithm, calculate the configuration parameters, and feed back the configuration parameters to the decellularization processing module.

[0168] To implement the above technical solution, in one embodiment, a decellularization device based on real-time data acquisition and process control implemented according to the above method is as follows: Figure 4 and Figure 5 As shown, it consists of three main modules:

[0169] The first module is the decellularization processing module 41, which includes a decellularization working solution classification and storage device 411, a liquid delivery unit 412 (which may be a precision CNC liquid delivery pump), a control unit 414 (which may be a CNC pump integrated controller), a decellularization processing container 413 (which may be a double-port decellularization processing container), and a decellularization controller 415 (which may be a bidirectional temperature-controlled decellularization process controller).

[0170] Figure 5 A structural diagram illustrating another embodiment of the decellularization device based on real-time data acquisition and process control according to an embodiment of the present invention is shown. Figure 5 As shown, compared to Figure 4The decellularization processing module 41 can be made up of one or more of the following: waste liquid collection unit 416 (which can be a waste liquid collection tank), liquid extraction unit 417 (which can be a precision CNC liquid extraction pump), and filter 418 (which can be a membrane filter).

[0171] It is worth noting that decellularization processing containers can be designed as follows: Figure 4 and Figure 5 The double-port shape shown can also be replaced with other processing containers with inlets and outlets.

[0172] The second module is the sample detection and data acquisition module 42, which includes an optical detector 421, and the optical detector 421 contains a multi-channel microfluidic chip 4211.

[0173] The third module is the data analysis and control system module 43, which includes a data storage server 431 and a control computer 432.

[0174] The decellularization module 41 is mainly used to automatically implement the decellularization process and automatically extract decellularization working solution samples. The decellularization working solution classification and storage unit 411 consists of multiple independent storage tanks for storing various types of decellularization working solutions (such as: SDS (Sodium Dodecyl Sulfate) solution, Triton-X 100 solution, DNase solution, PBS (Phosphate Buffered Sodium Chloride) solution). Each reservoir (containing saline, phosphate buffer, etc.) is connected to an independent precision CNC pump. When a sub-step in the decellularization process begins, the CNC pump integrated controller 414 delivers the decellularization working solution to the upper inlet of the dual-port decellularization processing container 413 as needed, according to the decellularization process settings (required decellularization working solution type and volume). The decellularization working solution and dECM material interact fully under the control of the bidirectional temperature-controlled decellularization process controller 415 to perform tissue and organ decellularization. The precision CNC pump 417 extracts a small amount of decellularization working solution through the lower outlet of the dual-port decellularization processing container 413 according to the settings. After filtration by the membrane filter 418, the solution is delivered to the next system module for sample detection. After the current process step is completed, all the decellularization working solution in the dual-port decellularization processing container 413 is extracted to the waste liquid collection tank, triggering the start of the next process step. The sample detection and data acquisition module 42 is mainly used to complete the detection and data acquisition of DNA and important ECM macromolecules in the decellularized working solution. The optical detector 421 contains a multi-channel microfluidic chip 4211. The chip is made of a highly transparent material with low adsorption of nucleic acid / protein molecules and is used for sample mixing, detection, and cleaning of the decellularized working solution.

[0175] The data analysis and overall control system module 43 is mainly used to collect and store real-time feedback information on the decellularization process and to control the implementation of the decellularization process. The data storage server 431 stores the sample data collected in real time by the previous system module and establishes or expands the dataset. At the same time, the overall control computer 432 solves and fits the curve, and then judges the current decellularization process effect based on the intelligent decision model established by the machine learning algorithm and optimizes the next process step. The system sends program instructions to the CNC pump integrated controller 414 in the decellularization processing module 41 to implement the next process step and realize closed-loop control.

[0176] Figure 6 A microfluidic chip structure for decellularization processing according to an embodiment of the present invention is shown. Figure 6 As shown, the microfluidic chip 4211 used for decellularization can be divided into:

[0177] Working fluid inlet 61 is used to receive the decellularized working fluid into the chip;

[0178] The detection solution inlet 62 is used to receive the decellularized detection solution into the chip;

[0179] The mixing unit 63 is connected to the working solution inlet and the detection solution inlet, and is used to mix the decellularized working solution and the decellularized detection solution;

[0180] The sample reading unit 64 is connected to the mixing unit and is used to read sample information.

[0181] In one embodiment, the microfluidic chip 4211 for decellularization further includes:

[0182] Waste liquid pool 65 is connected to the sample reading unit and is used to discharge waste liquid after reading sample information.

[0183] In one embodiment, the microfluidic chip 4211 for decellularization further includes:

[0184] The cleaning fluid inlet 66 is connected to the mixing channel and is used for the cleaning fluid to enter the chip.

[0185] Specifically, the working fluid inlet and the detection fluid inlet can be any device with an import function. The mixing unit can be a mixing channel or any device that achieves a mixing effect through methods such as shaking or centrifugation. The microfluidic chip 4211 used for decellularization can also be a multi-channel microfluidic chip.

[0186] In one embodiment, a trace amount of decellularized working solution sample extracted by the precision CNC pump 417 in the decellularization processing module 41 enters through the working solution inlet 61 of the multi-channel microfluidic chip 4211. After being thoroughly mixed with the detection solution entering through the detection solution inlet 62 of the multi-channel microfluidic chip 4211 in the mixing channel 63, the sample is detected and data is read by the optical detector 421 in the sample reading area 64. After reading, the liquid is discharged through the waste liquid pool 65. Subsequently, the cleaning solution enters the chip through the cleaning solution inlet 66, flows through the mixing channel 53 and the sample reading area 64, and is discharged through the waste liquid pool 65, completing the chip cleaning.

[0187] In one implementation, such as Figure 6 As shown, one or both of the waste liquid pool 65 and the cleaning fluid inlet 66 (within the dashed box) can be separated from the entire microfluidic chip 4211 used for decellularization. That is, when the waste liquid pool 65 is removed, the waste liquid can be discharged outside the microfluidic chip 4211 used for decellularization. When the cleaning fluid inlet 66 is removed, the cleaning fluid can enter the microfluidic chip 4211 through the working fluid inlet 61 or the detection fluid inlet 62.

[0188] Experimental verification

[0189] Experiments show that the intelligent and precise decellularization method based on real-time monitoring and feedback regulation described in this invention has the following advantages:

[0190] (1) Breakthrough in real-time quantitative and accurate detection of dECM material components, improving product stability and production efficiency. The content of DNA and important ECM macromolecules in the decellularization working solution is accurately determined using methods such as fluorescence staining and colorimetry. An indirect calculation method overcomes the problem of not being able to monitor the content of relevant components of dECM materials in real time during the decellularization process, achieving a transition from qualitative to quantitative analysis. The feedback data has high precision (up to 10 ng / mL), and the theoretical calculation values ​​of the content of relevant components of dECM materials are highly close to the actual measured values ​​(error only 5-10%). This can be effectively used to guide the optimization of the decellularization process, ensuring stable component content in different batches of dECM materials and improving product stability (see details). Figure 7A and Figure 7B ).

[0191] Figure 7A and Figure 7B A comparison chart of theoretical calculation and actual measurement errors according to an embodiment of the present invention is shown. Figure 7A In the diagram, the horizontal axis represents the time progress of decellularization treatment, the vertical axis represents the DNA content in the dECM material, and the upper and lower dashed lines represent the DNA content of natural porcine dermal skin and the risk standard, respectively. It can be seen that over time, the DNA content in the dECM material gradually decreases below the risk standard.

[0192] Figure 7B In the data, there are two sets of data: the solved value and the measured value. The two horizontal lines represent the mean of the results. It can be seen that the error between the measured value and the solved value obtained by using the technical solution of this invention is very small, and the stability of the product can be guaranteed.

[0193] Meanwhile, based on the trend of the fitted curve, the timeliness of the decellularization process can be determined. By reducing the non-time-sensitive treatment (the plateau period of the curve), the decellularization efficiency can be significantly improved. Taking porcine dermal dECM as an example, compared with the traditional decellularization process, using the method described in this invention, the decellularization processing time can be reduced from 66 hours to 27 hours, improving efficiency by nearly 60% (see details). Figure 8A and Figure 8B ).

[0194] Figure 8A and Figure 8B The images shown are the decellularization effects of the traditional process and the present invention, respectively. The horizontal axis represents the decellularization processing time, and the vertical axis represents the decellularization process. Figure 8A In traditional methods, two rectangular boxes can be seen indicating non-time-sensitive processing curve plateau areas, while Figure 8B After adopting the technical solution of this patent, the decellularization process presents a smooth curve with no obvious non-time-dependent treatment (curve plateau phase), and the decellularization efficiency is significantly improved.

[0195] (2) This method can distinguish the effects of DNA on fluorescence intensity from components of the decellularization working solution such as organic solvents, dissociation salts, and enzymes. Therefore, it can separately determine the residual amounts of DNA and toxic substances in dECM materials, thereby improving the safety of dECM materials by optimizing the decellularization process. Using the method of this invention, it can be ensured that the DNA content in different batches of dECM materials (more than 10 batches) is all below the risk standard (<50 ng / mg), effectively removing immunogenic substances (see details). Figure 9 Furthermore, dECM materials (free of toxic biochemical reagents) that are deemed safe by this method must maintain a cell viability of no less than 90% for more than 7 days in cell embedding experiments (see details). Figure 10 ).

[0196] Figure 9 The diagram illustrates the effect of removing immunogenic substances according to an embodiment of the present invention. Figure 9The image shows a comparison of the effects of staining different components in porcine dermal dECM and porcine dermal tissue. The first row shows the results of treating different cell nuclei, collagen, and glycosaminoglycans in porcine dermal dECM with different colored staining agents; the second row shows the results of treating different cell nuclei, collagen, and glycosaminoglycans in porcine dermal tissue with different colored staining agents. Compared with porcine dermal tissue, it can be seen that the porcine dermal dECM treated according to the embodiments of the present invention does not contain cell nuclei (hematoxylin-eosin staining), but it retains collagen (Mason staining) and glycosaminoglycans (Alcian blue staining) well, demonstrating that immune substances are completely removed and the molecular components and structure of ECM are fully preserved.

[0197] Figure 10 A graph showing the toxicity test results according to an embodiment of the present invention is provided. Figure 10 The image shows the number of viable cells measured at different time points after decellularization treatment. Figure 10 As can be seen, when human dermal fibroblasts are embedded in porcine dermal dECM hydrogel and cultured in three dimensions, the number of surviving cells (green) remains above 90% and is further increased on the seventh day, while almost no dead red cells are seen. This proves that the dECM material treated according to the embodiments of the present invention is non-toxic and safe for cells.

[0198] (3) The automated decellularization process is carried out using the method designed in this invention. The minimum data acquisition interval is 1 minute, which can effectively collect a large amount of data information reflecting the decellularization process parameters and process effects. Taking porcine dermal dECM as an example, based on the optimized decellularization process of 20 hours, a sample is taken every 5 minutes. Each material preparation can collect 240 sets of sample data, which is far superior to the efficiency of 1 set of data acquisition per time of the traditional method. Therefore, this method can be effectively used to establish mathematical models and intelligent decision-making algorithms for decellularization processes.

[0199] The functions of each module in each device of the embodiments of the present invention can be found in the corresponding descriptions in the above methods, and will not be repeated here.

[0200] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0201] Any process or method description in the flowchart or otherwise herein can be understood as representing a module, segment, or portion of code comprising one or more executable instructions for implementing a particular logical function or process, and the scope of the preferred embodiments of the invention includes additional implementations in which functions may be performed not in the order shown or discussed, including substantially simultaneously or in reverse order depending on the functions involved, as will be understood by those skilled in the art to which embodiments of the invention pertain.

[0202] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A decellularization method based on real-time data acquisition and process control, characterized in that, include: Obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be processed to form a basic reference dataset; Start the decellularization process according to the specified configuration parameters; At the preset decellularization treatment time points, samples were taken from the decellularization working solution to collect and measure the DNA and specified decellularization matrix ECM molecule content data. The DNA and specified decellularized matrix ECM molecule content data are fitted in real time to obtain the real-time fitting result, which is then compared with the basic reference dataset to obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution. Based on the real-time fitting results and / or the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution, the current progress of the decellularization process is quantitatively calculated to obtain decellularization process information. Feedback is provided based on the decellularization process information, and the specified configuration parameters during the decellularization process are adjusted in real time. The decellularization process ends when the progress stabilizes or a specified target is reached.

2. The method according to claim 1, characterized in that, The characteristic parameters include quality parameters.

3. The method according to claim 1, characterized in that, The characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated are obtained to form a basic reference dataset, including: The concentrations of DNA and specified decellularized matrix ECM molecules in the tissue to be treated, as well as the mass change rate of the lyophilized tissue to be treated, were determined. Based on the mass change rate, the actual mass of DNA and specified decellularized matrix ECM molecules contained in the tissue to be treated is calculated to form the basic reference dataset.

4. The method according to claim 1, characterized in that, At preset decellularization time points, samples are taken from the decellularization working solution to collect and measure the content data of DNA and specified decellularized matrix ECM molecules. The DNA and specified decellularized matrix ECM molecule content data are then fitted in real time to obtain the real-time fitting result, which is compared with the baseline reference dataset to obtain the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularization working solution, including: At preset time intervals, the decellularized working solution is sampled to obtain working samples. The working samples are then mixed with reactants to label DNA molecules and / or the specified decellularized matrix ECM molecules, resulting in a labeled mixture. Based on the aforementioned basic reference dataset, the actual concentration of DNA and specified decellularized matrix ECM molecules in the decellularized working solution is obtained through the characteristic parameters of the labeled mixture. The actual mass of DNA and specified decellularized matrix ECM molecules contained in the decellularized working solution is calculated based on the actual concentration and volume of the decellularized working solution, thus forming characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularized working solution.

5. The method according to claim 1, characterized in that, Based on the real-time fitting results, and / or the characteristic parameters of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularization working solution, the current progress of the decellularization process is quantitatively calculated, and the decellularization process information is obtained, including: At preset time intervals, the actual mass of DNA and specified decellularized matrix ECM molecules in the tissue to be treated and the decellularized working solution is fitted. In the fitting process, through formula M T / M P *100% quantitative calculation of the current progress of the decellularization process, where M T M represents the actual mass of DNA or a specified decellularized matrix ECM molecule in the decellularized working solution. P The actual mass of DNA or specified decellularized matrix ECM molecules in the tissue to be treated; By using interpolation, the actual mass of DNA and specified decellularization matrix (ECM) molecules in the tissue to be treated and the decellularization working solution are compared to obtain decellularization process information.

6. The method according to claim 1, characterized in that, Feedback is provided based on the decellularization process information, and the configuration parameters during the decellularization process are adjusted in real time, including: Based on the decellularization process information, determine the effectiveness of the decellularization process and the residual amount of the specified reagent; The actual mass of DNA and specified decellularized matrix ECM molecules is input into the decellularized working solution. A decision algorithm is constructed based on the progress level using a machine learning algorithm, and the configuration parameters are trained to obtain the configuration parameters. Adjust the decellularization process according to the configuration parameters.

7. A decellularization device based on real-time data acquisition and process control, characterized in that, include: A decellularization module is used to complete the decellularization process and extract decellularized working solution samples according to real-time updated configuration parameters; wherein, the decellularization module includes: A decellularized working solution classification and storage device, consisting of at least one independent storage tank, is used to store different types of decellularized working solutions; Liquid delivery unit, each of the liquid delivery units is connected to a corresponding liquid storage tank for delivering decellularized working solution; A decellularization processing container, connected to the liquid delivery unit, is used to receive the decellularization working solution delivered by the liquid delivery unit and to perform decellularization processing. A control unit, connected to the liquid delivery unit, is used to control the liquid delivery unit to deliver the decellularization working solution to the decellularization processing container according to the decellularization configuration parameters. The decellularization controller is used to regulate the decellularization process according to the specified decellularization processing configuration parameters; The sample detection and data acquisition module is used to perform real-time detection and data acquisition of characteristic parameters of DNA and specified decellularized matrix ECM molecules in the decellularization working solution, quantify the progress of the current decellularization process, and generate decellularization process information; wherein, the sample detection and data acquisition module includes: The calibration and detection unit includes at least one microfluidic chip for mixing, detecting, and cleaning the decellularized working solution, and transmitting the detection results to the data analysis and overall control system module in real time. The data analysis and overall control system module is used to analyze and store the real-time feedback information on the decellularization process and to control the decellularization process.

8. The apparatus according to claim 7, characterized in that, The sample detection and data acquisition module also includes: The liquid extraction unit is used to extract a specified volume of decellularized working solution from the outlet of the decellularization processing container according to specified configuration parameters.

9. The apparatus according to claim 8, characterized in that, The sample detection and data acquisition module also includes: A filter is used to filter the decellularized working solution extracted by the extraction unit.

10. The apparatus according to claim 7, characterized in that, The sample detection and data acquisition module also includes: Waste liquid collection unit, used to collect the waste liquid generated during the decellularization process.

11. The apparatus according to claim 7, characterized in that, The data analysis and overall control system module includes: The data storage unit is used to store the sample data collected in real time by the sample detection and data acquisition module, and to establish or update the decellularization process information. The control and computing unit is used to solve and / or fit, and to establish a decision model based on machine learning algorithms, calculate configuration parameters, and feed back the configuration parameters to the decellularization processing module.

12. A decellularization microfluidic chip for use in the sample detection and calibration unit of the decellularization device of claim 7, characterized in that, The microfluidic chip is made of a low / non-adsorbent material and is suitable for the decellularization method based on real-time data acquisition and process control as described in any one of claims 1-6, including: A working fluid inlet is provided to allow the decellularized working fluid to enter the chip. The detection solution inlet is used to receive the decellularized detection solution into the chip; A mixing unit, connected to the working solution inlet and the detection solution inlet, is used to mix the decellularized working solution and the decellularized detection solution; The sample reading unit, connected to the mixing unit, is used to read sample information.

13. The microfluidic chip according to claim 12, characterized in that, The microfluidic chip also includes: The cleaning fluid inlet is connected to the mixing unit and is used for the cleaning fluid to enter the chip.

14. The microfluidic chip according to claim 12, characterized in that, The microfluidic chip also includes: The waste liquid pool is connected to the sample reading unit and is used to discharge waste liquid after reading the sample information.

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