Cytotoxicity detection method based on micro-filtration membrane dynamic extraction

Abstract

The invention relates to the technical field of biological evaluation of medical instruments, in particular to a microfiltration membrane dynamic extraction-based cytotoxicity detection method which comprises the following steps: preparing a particle-containing gel sample to be detected into an extraction system; constructing a non-contact dynamic co-culture model containing a microfiltration membrane and cells; adding the leach liquor into the upper chamber, and carrying out dynamic leaching; cytotoxicity detection is carried out in the lower chamber. Particles are accurately intercepted through the micro-filtration membrane, detection errors caused by particle interference are avoided, the detection accuracy is improved, the authenticity of a detection result is ensured, 'false positive 'caused by particle interference in a traditional method is avoided, effective components of extract liquid are not affected by filtration, and a'micro-filtration membrane double-cavity dynamic co-culture system' is constructed; the slow release process of the material in tissue gaps after clinical injection is simulated, and dynamic monitoring of aging toxicity is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological evaluation of medical devices, and particularly to a cytotoxicity detection method based on dynamic extraction through a microfiltration membrane, which solves the problems of particle interference and component distortion in traditional methods through the dynamic extraction technology of the microfiltration membrane, and is applicable to the biocompatibility evaluation of class III high-risk injectable filling materials. Background Art

[0002] As a commonly used filling material in the field of medical aesthetics (such as facial wrinkle filling, tissue volume restoration), the safety of cross-linked sodium hyaluronate gel is directly related to the health of patients. According to the "Medical Device Classification Catalogue", this type of product belongs to class III high-risk medical devices and needs to strictly follow the GB / T 16886 series of standards for biological evaluation. However, the existing detection methods have the following defects: 1. High viscosity interference: Cross-linked particles or aggregates with a particle size > 1 μm in the gel are prone to physical sedimentation at the bottom of the well, resulting in mechanical damage or proliferation inhibition of adherent cells (such as L929 fibroblasts), leading to false positive results (such as misjudgment of dead cells by trypan blue staining); 2. Defects in static extraction: In the traditional extraction solution method (GB / T 16886.5-2017), the extraction solution is directly added to the cell well, which cannot simulate the continuous release process at the "material-tissue interface" after clinical injection, and the high-viscosity matrix may encapsulate toxic components, masking the true cytotoxicity; 3. Deviation in filtration pretreatment: Although removing particles through traditional centrifugation or filtration can avoid physical interference, it may adsorb or retain small molecule toxic components (such as cross-linking agent residues, degradation products), resulting in changes in the composition of the extraction solution, affecting the detection accuracy, and further verification is required to determine whether the leachables in the filtered extraction solution are still representative, and the steps are relatively cumbersome.

[0003] The prior art has not solved the three core problems of "particle barrier-component retention-dynamic contact" of high-viscosity materials, and there is an urgent need to develop a dedicated detection method to fill the industry gap. Summary of the Invention

[0004] The purpose of the present invention is to provide a cytotoxicity detection method based on dynamic extraction through a microfiltration membrane, aiming to solve the deficiencies in the cytotoxicity detection of high-viscosity cross-linked sodium hyaluronate gel in the prior art, especially the problems of particle interference, component distortion, and the inability of static extraction to simulate the actual clinical use scenario.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: A cytotoxicity detection method based on dynamic extraction through a microfiltration membrane, comprising the following steps: (1)Sample preparation: Add the gel to be tested to the MEM medium containing 10% fetal bovine serum at a ratio of 0.2 g / mL, and oscillate and extract it at 37 °C and 5% CO2 for 24 h to form an extract system containing particles; (2)Cell seeding: Seed the cells at a density of 1×10 5 cells / mL in a 24-well culture plate, with a predetermined volume of 1 mL per well, and culture for 24 h until near confluence; (3)Preparation of the microfiltration membrane positioning device: Fix the microfiltration membrane with a customized annular polyetheretherketone (PEEK) stent, place the stent with the fixed microfiltration membrane into the culture well of the 24-well culture plate, with a distance of 0.8 - 1.2 mm between the surface of the microfiltration membrane and the bottom of the culture well. The upper chamber is above the microfiltration membrane, and 0.5 mL of the particle-containing extract is added. The lower chamber is below the microfiltration membrane and is the cell layer inoculated with cells. The upper and lower chambers form a double-chamber culture system of "particle barrier - molecular permeability"; (4)Cell observation: Place the 24-well culture plate in an incubator, set the incubator parameters to 37 °C and 5% CO2, and culture for 48 h. Observe the cell morphology through a phase contrast microscope every 12 h during this period; (5)Cell cytotoxicity detection: Qualitative detection: Collect the cells in the lower chamber, stain them with 0.4% trypan blue for 5 min, count the proportion of unstained cells, and calculate the cell survival rate (%) = (number of live cells / total number of cells) × 100%; Quantitative detection: Discard the culture medium in the lower chamber, add 50 μL of MTT reagent to each well, incubate for 2 h, and then measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm), and calculate the relative growth rate (RGR) = (OD value of the test group / OD value of the medium control group) × 100%, where the medium control is the culture medium without the sample.

[0006] Furthermore, the microfiltration membrane is a hydrophilic polycarbonate microfiltration membrane, with a pore size of 3 - 10 μm, a thickness of 80 - 120 μm, and the surface is modified by hydroxylation treatment.

[0007] Furthermore, the cells are L929 mouse fibroblasts or other verified cells.

[0008] Furthermore, the viscosity range of the extract is 500 - 2000 mPa・s.

[0009] Furthermore, it also includes a component verification step for detecting key toxic components in the extract in the lower chamber.

[0010] Furthermore, the component verification step is to detect the residual amounts of hyaluronic acid oligosaccharides and BDDE cross-linking agent in the extract in the lower chamber by high performance liquid chromatography (HPLC) to ensure that the microfiltration membrane does not adsorb key toxic components (recovery rate ≥ 90%).

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. Solve the technical pain point of detection error caused by particle interference: Precisely intercept particles through a hydrophilic microfiltration membrane with a pore size of 3 - 10 μm, while allowing small molecule toxic components to pass through freely, isolating physical interference while retaining bioactive components, and ensuring the authenticity of the detection results.

[0012] 2. Simulate the dynamic toxicity release of the actual clinical use scenario: Construct a "microfiltration membrane double-chamber dynamic co-culture system", drive the continuous diffusion of small molecule components through the concentration difference on both sides of the membrane, simulate the slow release process of the material in the tissue space after clinical injection, and realize the dynamic monitoring of time-dependent toxicity, filling the gap in the evaluation of sustained-release materials by static detection.

[0013] 3. Establish a standardized detection method for high-risk materials: Provide a standardized detection process that meets the requirements of GB / T 16886.5 - 2022. By limiting the pore size of the microfiltration membrane, the distance between the membrane and the bottom of the well, and the combination of multi-index detections, form a repeatable and verifiable detection system, providing a scientific basis for medical device registration, quality control, and industry standard formulation.

[0014] 4. Ensure the accuracy of biocompatibility evaluation of high-risk materials: Through surface modification of the hydrophilic microfiltration membrane and non-contact culture design, ensure that the recovery rate of small molecule components in the leaching solution is ≥90%, avoid adsorption or interception of components by filtration, and truly reflect the cytotoxic effect of the extractable components of the material, providing reliable data support for the safety evaluation of Class III medical devices.

[0015] 5. By precisely detecting toxic components, the present invention can avoid the "false positives" caused by particle interference in traditional methods, ensure the clinical use safety of Class III medical devices, and will not affect the effective components of the leaching solution due to filtration, resulting in insufficient evaluation of the product, improving the detection accuracy. This method provides scientific support for the establishment of a new method for in vitro cytotoxicity evaluation of cross-linked sodium hyaluronate gel, and has a positive significance for the application safety of cross-linked sodium hyaluronate gel and its related medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the detection process of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] Example: As Figure 1 , a cytotoxicity detection method based on dynamic extraction with a microfiltration membrane, comprising the following steps: (1) Sample preparation: Add the gel to be tested to the MEM medium containing 10% fetal bovine serum at a ratio of 0.2 g / mL, and oscillate and extract for 24 h at 37 °C and 5% CO2 to form an extract system containing particles; (2) Cell seeding: Seed the cells at a density of 1×10 5 cells / mL in a 24-well culture plate, with a predetermined volume of 1 mL per well, and culture for 24 h until nearly confluent; (3) Prepare a microfiltration membrane positioning device: Fix the microfiltration membrane with a customized annular polyetheretherketone (PEEK) stent, place the stent with the fixed microfiltration membrane into the culture well of a 24-well culture plate, with a distance of 0.8 - 1.2 mm between the surface of the microfiltration membrane and the bottom of the culture well. The upper chamber is above the microfiltration membrane, and 0.5 mL of the extract containing particles is added. The lower chamber is below the microfiltration membrane, and the lower chamber is the cell layer inoculated with cells. The upper and lower chambers form a "particle barrier - molecular permeation" double-chamber culture system; By constructing a "microfiltration membrane double-chamber dynamic co-culture system", the continuous diffusion of small molecule components is driven by the concentration difference on both sides of the membrane, simulating the slow release process of the material in the tissue space after clinical injection, realizing the dynamic monitoring of time-dependent toxicity, and filling the deficiency of static detection in the evaluation of sustained-release materials. (4) Cell observation: Place the 24-well culture plate in an incubator, set the incubator parameters to 37 °C and 5% CO2, and culture for 48 h. Observe the cell morphology through a phase contrast microscope every 12 h during this period; (5) Cytotoxicity detection: Qualitative detection: Collect the cells in the lower chamber, stain with 0.4% trypan blue for 5 min, count the proportion of unstained cells, and calculate the cell survival rate (%) = (number of live cells / total number of cells) × 100%; Quantitative detection: Discard the culture medium in the lower chamber, add 50 μL of MTT reagent to each well, incubate for 2 h, and then measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm), and calculate the relative growth rate (RGR) = (OD value of the test group / OD value of the medium control group) × 100%, where the medium control is the culture medium without the sample.

[0019] The microfiltration membrane is a hydrophilic polycarbonate microfiltration membrane with a pore size of 3 - 10 μm and a thickness of 80 - 120 μm. The surface is treated with hydroxylation modification, which can effectively retain gel particles with a particle size > 5 μm (retention rate > 95%), and at the same time allow the degradation products of hyaluronic acid (oligosaccharides, cross-linker fragments) to pass freely, isolating physical interference while retaining bioactive components to ensure the authenticity of the detection results.

[0020] The cells are L929 mouse fibroblasts or other verified cells.

[0021] The viscosity range of the leaching solution is 500 - 2000 mPa・s.

[0022] It also includes a component verification step for detecting key toxic components in the lower chamber leaching solution.

[0023] The component verification step is to detect the residual amounts of hyaluronic acid oligosaccharides and BDDE crosslinker in the lower chamber leaching solution by high performance liquid chromatography (HPLC) to ensure that the microfiltration membrane does not adsorb key toxic components (recovery rate ≥ 90%).

[0024] The specific operation process of a cytotoxicity detection method based on dynamic leaching of a microfiltration membrane: 1. Microfiltration membrane system construction and sample pretreatment Membrane parameter design: Select a hydrophilic polycarbonate microfiltration membrane with a pore size of 3 - 10 μm (preferably 3 - 5 μm) (the surface is hydroxylated modified, water flux ≥ 500 mL / cm²・h), a thickness of 80 - 120 μm, effectively retaining gel particles with a particle size > 5 μm, while allowing hyaluronic acid degradation products (oligosaccharides, crosslinker fragments) to pass freely.

[0025] Sample preparation: Add the gel to be tested to MEM medium containing 10% fetal bovine serum at a ratio of 0.2 g / mL, and oscillate and leach for 24 h at 37°C and 5% CO2 to form a leaching solution system containing particles (viscosity range 500 - 2000 mPa・s).

[0026] 2. Non-contact dynamic co-culture model Cell seeding: Seed L929 mouse fibroblasts at a density of 1×10 5 cells / mL into the wells of a 24-well culture plate, with a volume of 1 mL per well, and culture for 24 h until nearly confluent.

[0027] Microfiltration membrane positioning device: Use a customized annular polyether ether ketone (PEEK) bracket to fix the microfiltration membrane, ensuring that the distance between the membrane surface and the bottom of the well is 1.0 ± 0.1 mm. Add 0.5 mL of the particle-containing leaching solution to the upper chamber and the cell layer to the lower chamber to form a "particle barrier - molecular permeation" double-chamber culture system.

[0028] 3. Dynamic leaching and toxicity detection Culture conditions: Place the 24-well culture plate in an incubator for culture. Set the incubator parameters to 37°C and 5% CO2, and culture for 48 h. During this period, observe the cell morphology (such as cell membrane integrity, pseudopod extension, and change in nuclear-cytoplasmic ratio) through a phase contrast microscope every 12 h.

[0029] Qualitative detection: Trypan blue test: Collect the cells in the lower chamber, stain with 0.4% trypan blue for 5 min, count the proportion of unstained cells, and calculate the cell survival rate (%) = (number of live cells / total number of cells) × 100%.

[0030] Quantitative detection: MTT method: After discarding the culture medium, add 50 μL of MTT reagent to each well, incubate for 2 h, and then measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm). Calculate the relative growth rate (RGR) = (OD value of the test group / OD value of the medium control group) × 100%, where the medium control is the culture medium without the sample.

[0031] Component verification: Detect the residual amounts of hyaluronic acid oligosaccharides and BDDE crosslinking agent in the extract of the lower chamber by high performance liquid chromatography (HPLC) to ensure that the microfiltration membrane does not adsorb key toxic components (recovery rate ≥ 90%).

[0032] By limiting the pore size of the microfiltration membrane (3 - 10 μm), the distance between the microfiltration membrane and the bottom of the well (0.8 - 1.2 mm), and a combination of multiple index detections (trypan blue staining for qualitative detection + MTT method for quantitative detection + HPLC component verification), the present invention forms a repeatable and verifiable detection system, providing a scientific basis for the registration, quality control, and industry standard formulation of medical devices.

[0033] Through the surface modification of the hydrophilic microfiltration membrane (hydroxylation treatment, water flux ≥ 500 mL / cm²·h) and non-contact culture design, the present invention ensures that the recovery rate of small molecule components (such as crosslinking agents and oligosaccharides with molecular weight < 10 kDa) in the extract is ≥ 90%, avoids the adsorption or retention of components by filtration, truly reflects the cytotoxic effect of the extractable substances of the material, and provides reliable data support for the safety evaluation of class III medical devices.

[0034] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A cytotoxicity detection method based on dynamic extraction with a microfiltration membrane, characterized in that It includes the following steps: (1) Sample preparation: Add the gel to be tested into the MEM medium containing 10% fetal bovine serum at a ratio of 0.2 g / mL, and oscillate and extract at 37 °C and 5% CO2 for 24 h to form an extract system containing particles; (2) Cell seeding: Seed the cells at a density of 1×10 5 cells / mL into a 24-well culture plate, with a predetermined volume of 1 mL per well, and culture for 24 h until near confluence; (3) Preparation of the microfiltration membrane positioning device: Fix the microfiltration membrane using a custom-made annular polyetheretherketone (PEEK) stent, place the stent with the fixed microfiltration membrane into the culture wells of a 24-well culture plate, with a distance of 0.8 - 1.2 mm between the surface of the microfiltration membrane and the bottom of the culture well. The upper chamber is above the microfiltration membrane, and 0.5 mL of the extract containing particles is added. The lower chamber is below the microfiltration membrane and is the cell layer seeded with cells. The upper and lower chambers form a double-chamber culture system of "particle barrier - molecular permeability"; (4) Cell observation: Place the 24-well culture plate in an incubator with the incubator parameters set at 37°C and 5% CO2, and culture for 48 h. Observe the cell morphology through a phase contrast microscope every 12 h during this period; (5) Cytotoxicity detection: Qualitative detection: Collect the cells in the lower chamber, stain with 0.4% trypan blue for 5 min, count the proportion of unstained cells, and calculate the cell survival rate (%) = (number of live cells / total number of cells) × 100%; Quantitative detection: Discard the culture solution in the lower chamber, add 50 μL of MTT reagent to each well, measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm) after incubation for 2 h, and calculate the relative growth rate (RGR) = (OD value of the test group / OD value of the medium control group) × 100%, where the medium control is the medium without the sample.

2. The cytotoxicity detection method based on dynamic extraction with a microfiltration membrane according to claim 1, wherein: The microfiltration membrane is a hydrophilic polycarbonate microfiltration membrane with a pore size of 3 - 10 μm, a thickness of 80 - 120 μm, and the surface is treated by hydroxylation modification.

3. The cytotoxicity detection method based on dynamic extraction with a microfiltration membrane according to claim 1, wherein: The cells are L929 mouse fibroblasts.

4. The cytotoxicity detection method based on dynamic extraction with a microfiltration membrane according to claim 1, characterized in that: The viscosity range of the extract is 500 - 2000 mPa・s.

5. The cytotoxicity detection method based on dynamic extraction with a microfiltration membrane according to claim 1, characterized in that: It also includes a component verification step for detecting key toxic components in the lower chamber extract.

6. The cytotoxicity detection method based on dynamic extraction with a microfiltration membrane according to claim 5, wherein: The component verification step is to detect the residual amounts of hyaluronic acid oligosaccharides and BDDE cross-linking agent in the lower chamber extract by high performance liquid chromatography (HPLC) to ensure that the microfiltration membrane does not adsorb key toxic components.

Images