Improved in-situ culture method for removing maternal blood and impurity cell pollution in amniotic fluid

Abstract

The invention provides an improved in-situ culture method for removing maternal blood and impurity cell pollution in amniotic fluid, which focuses on key window period intervention: in a pre-culture stage (at a time point when fetal cells are sporadic adheres to the wall and maternal blood cells are not firmly adherent to the wall), a vertical dropwise addition flushing method is adopted; the non-wall-attached / infirmly-attached maternal blood impurities are physically removed by utilizing the impact force of the liquid flow, and repeated passage is not needed. And through a direct intervention means of physical flushing in a window period, the damage of passage to fetal cells is avoided, and the operation is more efficient. Besides, a combined scheme of in-situ culture and vertical dropwise addition flushing is designed for a bloody amniotic fluid sample, cell positions are fixed through a glass slide growth area of an in-situ culture box, and then accurate flushing is realized by vertical dropwise addition with the height of 3-5cm, so that fetal cells firmly adhered to the wall are reserved, and maternal blood impurities are removed.

Description

Technical Field

[0001] This invention relates to the field of bioculture technology, and in particular to an improved in situ culture method for removing maternal blood and impurity cell contamination from amniotic fluid and its application. Background Technology

[0002] In the field of prenatal diagnosis, amniotic fluid cell chromosome examination is an important method for diagnosing fetal chromosomal abnormalities (such as trisomy 21 and trisomy 18). Currently, the commonly used amniotic fluid cell culture method is in situ culture, where amniotic fluid cells are seeded onto a glass slide, and after the cells adhere and grow to form clones, they are directly harvested and analyzed for chromosomes. However, in clinical practice, in situ culture of bloody amniotic fluid often fails. Bloody amniotic fluid samples mainly include two situations: first, due to previous pregnancy-pregnancy treatment to maintain the pregnancy, old bleeding may occur in the uterine cavity, causing the amniotic fluid to appear brown, containing a large number of ruptured and degenerated maternal blood cells and their fragments; second, damage to maternal capillaries during amniocentesis leads to fresh blood mixing into the amniotic fluid. Whether due to old or fresh maternal blood contamination, the maternal blood cells and cell fragments cannot adhere and grow in the culture system, but will deposit and adhere to the culture surface, severely crowding out the adhesion space for fetal amniotic fluid cells and potentially releasing metabolic products that interfere with fetal cell growth, which is a major cause of culture failure.

[0003] According to the "Expert Consensus on Quality Control of Amniotic Fluid Cell In situ Culture Technology (2025 Edition)," successful preparation of chromosome karyotypes and issuance of reports typically requires harvesting at least 15 cells from 15 clones in two independent culture systems. Both of the aforementioned situations can lead to insufficient clone count for testing, resulting in culture failure, prolonged testing cycles, and even the need for repeat amniocentesis, increasing the risks and financial burden on pregnant women, while also affecting the reliability of the test results. Currently, the conventional treatment for the aforementioned contamination problem is to perform multiple passages. For example, the invention patent with publication number CN115058386B proposes a method and application for amniotic fluid cell culture to remove maternal blood cell contamination from bloody amniotic fluid (the patent content is as follows: S1, after solid-liquid separation, the supernatant of the bloody amniotic fluid is removed and mixed with amniotic fluid culture medium to obtain a cell suspension; the cell suspension is inoculated into a culture dish for culture to obtain a first culture; S2, the amniotic fluid culture medium is added to the first culture and culture is continued to obtain a second culture; S3, maternal blood cells and the original culture medium are removed from the second culture, and then the amniotic fluid culture medium is added to it and culture is continued to obtain a third culture; S4, the third culture is passaged to obtain a passaged culture; S5, fetal cells in the passaged culture are collected). This method indirectly removes maternal blood cells through the "passage screening" process of "centrifugation followed by mixing culture medium for culture → multiple medium changes for passage → collection of fetal cells", which still relies on cell screening during the passage process. These methods are inefficient, cumbersome, and multiple passages can increase the risk of cell damage and contamination, and are not a fundamental solution.

[0004] Therefore, there is an urgent need for a novel in situ amniotic fluid cell culture method that can simply and efficiently remove maternal blood cells and impurity cells from amniotic fluid. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides the following technical solution:

[0006] On the one hand, an improved in situ culture method for removing maternal blood and impurity cell contamination from amniotic fluid is provided, the method comprising the following steps:

[0007] S1: Sample processing and inoculation: After centrifuging the amniotic fluid sample, discard the supernatant and retain the cell pellet; resuspend the cell pellet using amniotic fluid culture medium to prepare a cell suspension, and inoculate the cell suspension into a culture container containing a growth surface;

[0008] S2: Initial adherence culture: The culture container after inoculation is placed in static culture at 37°C and 5% CO2 to allow the fetal cells in the amniotic fluid to initially adhere to the growth surface.

[0009] S3: Key flushing and adding of culture medium: After the initial adherence culture is completed, use a sterile pipette to draw fresh amniotic fluid culture medium and drop the culture medium vertically and stably onto the growth surface from a height of 3-5 cm directly above the sample well of the culture container. Use the physical impact force of the liquid flow to flush the growth surface to remove maternal blood cells and impurity cells that have not adhered or are not firmly attached.

[0010] S4: Medium change and passage: After continued culture, when more than 10 cell clones with a diameter greater than 2 mm appear on the growth surface, perform medium change or passage.

[0011] S5: Cell Harvesting: When the cell clones grow to meet the harvesting criteria, pure fetal amniotic fluid cells are harvested.

[0012] Preferably, in step S1, the centrifugation conditions are: room temperature, 1500 rpm, centrifugation for 10 minutes.

[0013] Preferably, in step S1, the volume of the inoculated cell suspension is 0.5-1 mL.

[0014] Preferably, in step S2, the initial adherent culture time is 40±n (n takes the value of 1-3) hours.

[0015] Preferably, in step S3, the critical rinsing and liquid addition operation is performed 40 hours after the initial adherence culture, when it is confirmed under a microscope that a few fetal cells have adhered to the culture vessel.

[0016] Preferably, in step S3, the fresh amniotic fluid culture medium is preheated to 37°C before use, and the volume of the fresh amniotic fluid culture medium added is 3±n (n takes the value of 1-2) mL.

[0017] Preferably, in step S3, after completing the key rinsing and adding of liquid, the culture container is returned to 37°C and 5% CO2 conditions for continued culture for 5±n (n takes the value of 1-3) days.

[0018] Preferably, in step S4, the liquid replacement operation specifically involves: after aspirating the old culture medium, moving the pipette tip to one end of the growth surface, and slowly and evenly rinsing the entire growth surface with fresh culture medium in a horizontal direction. This rinsing action is repeated twice.

[0019] Preferably, in step S5, the harvesting standard is: at least 15 cell-rich medium-to-large clones on the growth surface, wherein clones with a diameter ≥ 6 mm are large clones, and clones with a diameter of 2-6 mm are medium clones; the specific cell harvesting method is as follows:

[0020] (1) Colchicine treatment: Add colchicine solution to the culture container to make the final concentration about 100 µg / mL, and act on it for 30 minutes at 37℃ and 5% CO2.

[0021] (2) Hypotonic treatment: After aspirating the culture medium, add a mixture of 0.4% sodium citrate and 0.4% potassium chloride preheated to 37°C and let it stand at 37°C for 30 minutes;

[0022] (3) Fixation and slide preparation: The slides were fixed three times in sequence with glacial acetic acid pre-fixation and methanol:glacial acetic acid fixation solution with a volume ratio of 3:1. Then the slides were dried in an environment of 25°C and 50% humidity and baked at 80°C for 3.5 hours.

[0023] (4) Banding: G-banded chromosome slides were prepared by digestion with trypsin and Giemsa staining.

[0024] On the other hand, an application is provided for pure fetal amniotic fluid cells harvested based on the amniotic fluid cell culture method described above, which can be used for chromosome preparation, karyotype analysis, or for collecting precipitates after digestion for molecular detection.

[0025] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following innovative effects:

[0026] 1. Timing Innovation: The study distinguished the differences in adhesion dynamics between fetal cells and contaminant cells in amniotic fluid cell culture, determined the optimal intervention time point, and eliminated contaminant interference while protecting the target cells.

[0027] 2. Methodological Innovation: This method proposes a simple physical operation—"rinsing with fresh culture medium"—in an in-situ culture system to replace complex chemical treatments or multiple passages, thereby achieving efficient removal of maternal blood cells and impurity cells. This method is ingeniously conceived, extremely low-cost, and remarkably effective.

[0028] 3. Process Innovation: The decontamination step is seamlessly integrated into the standard in-situ culture process, forming a new standardized process of "inoculation → short-term adhesion → rinsing and decontamination → routine culture". This process is an important and practical improvement over the traditional in-situ culture method.

[0029] 4. Application Innovation: This method directly solves a major clinical pain point that leads to amniotic fluid culture failure, significantly improves the success rate of culture of difficult samples (bloody), and ensures the sample quality and reliability of subsequent chromosome and molecular genetic testing. It has clear clinical value and market application prospects. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a flowchart illustrating the modified in situ culture method for removing maternal blood and impurity cells from amniotic fluid, as described in this application.

[0032] Figure 2 This is a schematic diagram of the key rinsing and liquid addition operation of this application, showing the relative position of the liquid flow direction, the drop height and the culture vessel when fresh culture medium is added vertically.

[0033] Figure 3 This is a schematic diagram comparing the cell state before and after rinsing and adding fluid. The top shows the state of maternal blood cells and impurities covering the growth surface before adding fluid, while the bottom shows the state after vertical dripping and rinsing, where impurities are removed and fetal amniotic fluid cells are retained.

[0034] Figure 4 This is a visual comparison of clonal growth observed under a fully automated chromosome scanner after 13 bloody amniotic fluid samples were treated with different fluid addition methods.

[0035] Figure 5 A bar chart comparing the number of effective clones in 13 bloody amniotic fluid samples in the routine fluid addition group and the vertical drip flushing group.

[0036] Figure 6 A line graph showing the trend of effective clones in 13 bloody amniotic fluid samples across two treatment groups reflects the trend of consistency changes among the samples.

[0037] Figure 7 A detailed comparison table of the effective clone counts in 13 bloody amniotic fluid samples across the two treatment groups is provided, listing the specific data for each sample and the statistical results between the groups. Detailed Implementation

[0038] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0039] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0040] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, their intended meanings are consistent. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, their intended meanings are consistent.

[0041] In this embodiment of the invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meaning they express is the same.

[0042] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0043] In this embodiment, the samples, reagents, or experimental equipment used can be provided by the laboratory or purchased commercially.

[0044] In this embodiment, the proposed approximation can fluctuate by ±5-10% from its example value, depending on the specific requirements.

[0045] The technical implementation scheme of this application will be described in detail below.

[0046] (a) Purpose of this application

[0047] 1. Solves the problem of maternal blood cell and impurity cell contamination: Effectively removes maternal blood cells and impurity cells deposited on the surface of the slide, making room for fetal amniotic fluid cells to adhere to the slide.

[0048] 2. Improve culture success rate and clone count: By removing physical barriers and biological inhibitions from contaminated cells, the number of adherent fetal cell clones is significantly increased, ensuring that the cloning standards required for chromosome analysis are met.

[0049] 3. Simplified operation process: It provides a convenient method that can remove contaminated cells in the early stage of culture without relying on multiple passages, shortening the culture cycle and reducing the complexity of operation and the risk of contamination.

[0050] 4. Improved accuracy and reliability of detection: Obtaining a higher purity fetal cell population avoids interference from maternal cell DNA, providing high-quality samples for subsequent high-throughput molecular detection such as chromosome karyotype analysis, SNP-array, and CNV-seq, and ensuring the accuracy of test results.

[0051] (II) Technical Solution of this Application

[0052] This invention provides a method for amniotic fluid cell culture to remove maternal blood cells and other contaminating cells from in situ culture. The core of this method is to physically remove non-adherent contaminating cells during the critical window period after cell seeding and adhesion but before the large-scale growth of clones, through a specific liquid addition and rinsing operation.

[0053] like Figure 1 As shown, the steps of this method are as follows:

[0054] S1: Sample processing and inoculation

[0055] (1) Collect amniotic fluid samples (usually 8-10 mL of bloody amniotic fluid) and centrifuge (1500 rpm, 10 min at room temperature) to achieve solid-liquid separation.

[0056] (2) Discard the supernatant and retain the cell precipitate.

[0057] (3) Add an appropriate amount of amniotic fluid culture medium (Amnio Type-1 Medium, VivaCell, 2422485, 100mL) to the cell pellet, resuspend and mix well to prepare a cell suspension.

[0058] (4) Inoculate the cell suspension (usually 0.5-1 mL) into a culture dish containing a glass slide.

[0059] S2: Initial adherent culture

[0060] (1) Place the inoculated culture box in a 37℃, 5% CO2 incubator for static culture for about 40 hours.

[0061] (2) The purpose of this stage is to allow the fetal cells in the amniotic fluid (mainly fibroblast-like cells and epithelial-like cells) to initially adhere to the glass slide.

[0062] S3: Key flushing fluid addition (the core improvement of this method)

[0063] (1) After about 40 hours of static culture, observation under a microscope confirmed that there were sporadic fetal cells attached to the wall.

[0064] (2) Perform the procedure inside a biosafety cabinet: Use a sterile pipette to draw approximately 3 mL of fresh, preheated amniotic fluid culture medium to 37°C. Place the pipette at a height of approximately 3-5 cm above the sample well in the culture box and add the culture medium vertically and steadily. Utilize the gentle impact of the falling liquid to gently irrigate the growth surface of the slide, such as... Figure 2 As shown.

[0065] (3) Rinsing principle and operation: Vertical dripping at this height can generate a moderate liquid flow, sufficient to wash away maternal blood cells, red blood cells and other impurity cells that have not adhered or are not firmly attached from the surface of the slide and suspend them in the culture medium. Fetal cells that have initially adhered to the slide are retained due to their stronger adhesion. The rinsing process is as follows: Figure 3 As shown.

[0066] (4) After the addition is complete, place the culture box back into the incubator at 37°C and 5% CO2 and continue culturing for about 5 days. During this period, the growth of cell clones can be observed under a microscope periodically.

[0067] S4: Fluid Change and Subculture

[0068] (1) Observe under a microscope to check the cell adhesion and morphology. When more than 10 clones larger than 2 mm appear (clones with a diameter ≥ 6 mm are large clones, those with a diameter of 2 ~ 6 mm are medium clones, and those with a diameter ≤ 2 mm are small clones), change the medium. Aspirate the culture medium from one side, then move the pipette tip to the starting position at the left end of the growth surface, and slowly and evenly rinse the entire cell growth surface from left to right in a horizontal direction. Repeat this rinsing action twice to further remove any small amount of impurities or metabolites that may detach, while avoiding impact on the adhered clones. Aspirate and inoculate into a new culture box, add 1 mL of amniotic fluid culture medium, and perform subculture.

[0069] (2) Add 3 mL of amniotic fluid culture medium to the original box and continue culturing for 1 day.

[0070] S5: Cell Harvesting and Chromosome Preparation

[0071] When the fetal cell clones on the slide grow to meet the harvest criteria (at least 15 large and abundant cell clones on two-line slides), harvesting is usually carried out around day 8 of culture. The obtained cells are purified fetal amniotic fluid cells, which can be used for chromosome preparation, karyotype analysis, or digestion and collection of the precipitate for molecular detection.

[0072] (III) Examples

[0073] 1. Experimental Design

[0074] Thirteen clinical bloody amniotic fluid samples were collected in 2025 (sample numbers 2025001-2025013, approximately 15-20 mL in volume). Under aseptic conditions, each sample was aliquoted into two equal portions (Group A and Group B). Both Group A and Group B underwent the same basic amniotic fluid cell in situ culture procedure, differing only in the crucial step of "liquid addition" to assess the impact of different addition methods on removing maternal blood cell contamination and promoting fetal cell clone formation.

[0075] Group A (control group): The conventional method of adding culture medium was used, that is, fresh culture medium was slowly added along the side wall of the culture box to avoid directly impacting the cell growth surface.

[0076] Group B (Experimental Group): The vertical dripping and rinsing method described in this invention was used, that is, fresh culture medium was added vertically from a certain height (3-5 cm) to gently rinse the cell growth surface.

[0077] Both samples were cultured independently until harvest, and the effectiveness was evaluated by comparing the number of effective cell clones (clones larger than 2 mm) that could ultimately be used for chromosome analysis.

[0078] 2. Experimental Methods

[0079] 2.1 Sample processing and inoculation (the procedures are the same for both Group A and Group B)

[0080] Take approximately 8-10 mL of aliquoted bloody amniotic fluid and place it in a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 10 minutes at room temperature. Carefully discard the supernatant, retaining approximately 0.5 mL of liquid. Add 1 mL of amniotic fluid culture medium (Amnio Type-1 Medium, VivaCell, 2422485, 100 mL) to each cell pellet, gently pipette to mix, and prepare a cell suspension. Seed the suspension into the growth areas of two independent standard in situ culture chambers, labeling them A and B. Smoothly transfer the culture chambers to an incubator at 37°C and 5% CO2 for pre-incubation. The pre-incubation period is 24 hours.

[0081] 2.2 Key liquid addition steps (different operations for groups A and B)

[0082] After 24 hours of pre-culture, observation under a microscope confirmed that there were sporadic fetal cells attached to the slides in both groups, while a large number of unattached maternal blood cells and impurities were also visible on the growth surface.

[0083] Group A (Routine Addition): In a biosafety cabinet, use a sterile pipette to draw 3 mL of fresh amniotic fluid culture medium. Place the pipette tip against the inner wall of the well in the culture chamber and slowly add the culture medium along the side wall, avoiding direct contact with the cell growth surface of the slide as much as possible. After adding the medium, return the chamber to the incubator.

[0084] Group B (Liquid addition method of this invention): In a biosafety cabinet, use a sterile pipette to draw 3 mL of fresh amniotic fluid culture medium, place the pipette tip vertically about 3-5 cm above the culture box, and steadily and vertically add the culture medium to the growth area of ​​the slide, using the gentle impact of the liquid flow to rinse the growth surface in all directions.

[0085] After adding the liquid, both groups A and B culture boxes were placed at 37℃ and 5% CO2 and cultured for another 5 days.

[0086] 2.3 Subculturing and Medium Change

[0087] Five days later, observe the cells under a microscope to check their adhesion and morphology. Change the medium, aspirate the culture medium from one side, gently wash the cell growth surface once, and then aspirate and inoculate into a new culture box. Add 1 mL of amniotic fluid culture medium for passage. Add 3 mL of amniotic fluid culture medium to the original box and continue culturing for 1 day.

[0088] 2.4 Cell Harvesting and Chromosome Preparation

[0089] On day 8 of culture, when the cell clones under a microscope meet the harvest criteria, the two groups of samples are harvested simultaneously.

[0090] Colchicine treatment: Add 60 μL of colchicine solution diluted 10 times with culture medium to each culture box (to make the final concentration approximately 100 μg / mL), and continue to treat at 37℃ and 5% CO2 for 30 minutes.

[0091] Hypotonic treatment: Discard the culture medium, add 5 mL of a hypotonic solution of 0.4% sodium citrate and 0.4% potassium chloride preheated to 37°C, and let stand at 37°C for 30 minutes.

[0092] Fixation and slide preparation: Pre-fixation with glacial acetic acid and three fixation with methanol:glacial acetic acid (3:1) fixative were performed according to standard procedures. The slides were removed and air-dried in a temperature- and humidity-controlled environment (25℃, 50%), and then baked at 80℃ for 3.5 hours.

[0093] Banding: G-banded chromosome slides were prepared by digestion with trypsin and Giemsa staining.

[0094] This department achieves efficient cell harvesting and high-quality chromosome preparation by precisely controlling the concentration of key reagents and process parameters. Colchicine is used at a final concentration of 100 μg / mL and an incubation time of 30 minutes, effectively blocking spindle formation to enrich the mitotic phase while avoiding the risk of chromosome breakage due to overtreatment. Hypotonic treatment uses a mixture of 0.4% sodium citrate and 0.4% potassium chloride (5 mL / sample), and a constant temperature of 37℃ promotes uniform cell membrane expansion, reducing chromosome clumping. The fixation process employs glacial acetic acid pre-fixation combined with three fixations using methanol:glacial acetic acid (3:1), followed by drying at 25℃ / 50% humidity and baking at 80℃ for 3.5 hours, significantly improving chromosome structural stability. The synergistic treatment of trypsin digestion and Giemsa staining yields clear G-banding bands, providing high-quality samples for subsequent karyotype analysis. The overall process balances cell harvesting efficiency with chromosome morphological integrity.

[0095] 2.5 Results Analysis and Comparison

[0096] The chromosome slides prepared in groups A and B were scanned using a fully automated chromosome scanner. The system automatically transmitted the data to a computer for further analysis. A direct comparison of clonal growth observed under the fully automated chromosome scanner was provided for 13 bloody amniotic fluid samples treated with different fluid addition methods. Figure 4 As shown in the bar chart, the effective clone counts in the conventional liquid addition group and the vertical drip flushing group are compared as follows: Figure 5 As shown, the line trend of the effective clone number in the two treatment groups is compared as follows: Figure 6 As shown in the table, a detailed comparison of the effective clone counts in the two treatment groups is as follows: Figure 7 As shown.

[0097] from Figure 4 It can be seen that samples treated with the conventional wall-feeding method have sparse distribution of effective clones on the growth surface of the slide, while the corresponding samples treated with the vertical drip-washing method described in this application have a greater number of effective cell clones, proving that this method is effective in removing contaminants and promoting fetal cell clone growth; at the same time, by Figure 5 The results showed that the average number of effective clones in the conventional fluid addition group (13 samples) was 14.7, while the average number of effective clones in the vertical drip-washing group reached 24.6, representing an average increase of approximately 67.3%. This statistical result quantitatively demonstrates that the vertical drip-washing method of this application can significantly increase the number of effective cell clones obtained after culturing bloody amniotic fluid samples. Figure 6 The line graphs show that for all 13 samples, the line representing the vertical drip rinsing group consistently lies above the line representing the conventional liquid addition group, with the two lines exhibiting a clear and consistent separation trend. This demonstrates that the effectiveness of this method is not an isolated case, exhibiting consistent validity and universality. Finally, the analysis... Figure 7 Specific data shows that after vertical drop-washing, the number of effective clones in all 13 samples was higher than that after conventional treatment, with the increase ranging from 3 to 27 clones. In the conventional liquid addition group, some samples (such as 2025005, 2025006, 2025007, and 2025009) had a low number of effective clones (<10), which did not meet the clone count requirement usually required for chromosome karyotype analysis (usually ≥10 clones), posing a risk of culture failure. However, after treatment with this method, the number of effective clones in these samples was increased to near or above the standard requirement, greatly improving the culture success rate and the reliability of the test results.

[0098] 3. Experimental Conclusions

[0099] In summary, this embodiment demonstrates through direct comparison that, in the in situ culture of bloody amniotic fluid, the vertical drip-washing method described in this invention, compared with the traditional slow-addition method along the wall, can efficiently remove maternal blood cells and impurities adhering to the growth surface, creating a superior growth environment for fetal amniotic fluid cells. This improves the success rate and efficiency of obtaining sufficient analytical sample clones in a single culture, proving the outstanding effect of this invention in solving the problem of bloody amniotic fluid culture and ensuring the reliability of chromosome karyotype analysis.

[0100] For the specific application and implementation of this solution, the following technical parameters can be selected by the user:

[0101] (1) Timing of rinsing: The key is to rinse after the fetal cells have adhered to the culture medium and before the contaminated cells have been fixed. The specific timing can be adjusted within the range of 20-48 hours after inoculation, and should be optimized according to different types of culture medium, serum content and cell activity.

[0102] (2) Rinsing method:

[0103] Basic procedure: Use a sterile pipette to draw fresh culture medium and gently and slowly drop it onto the surface of a glass slide, using the liquid flow to rinse it.

[0104] Alternative solution: Tilt the petri dish slightly to allow the liquid to flow from one end to the other, thus achieving a "flow-through" rinsing of the slide.

[0105] (3) Culture vessels: not limited to glass slides in standard culture boxes, but also applicable to in situ culture systems in which glass slides are placed in culture plates, or special culture bottles with growth surfaces.

[0106] (4) Sample type: In principle, this method is not only applicable to bloody amniotic fluid, but can also be used to treat amniotic fluid samples with a large number of impurity cells due to meconium contamination or other reasons, as well as to remove a large number of non-target cells in other body fluid cell cultures (such as pleural and peritoneal fluid).

[0107] (5) This application does not explicitly limit the amniotic fluid culture medium used, which can be any of the commercially available amniotic fluid culture media. In some specific embodiments, the amniotic fluid culture medium used is manufactured by VivaCell, with a production batch number of 2422485 and a specification of 100mL.

[0108] (6) This application does not explicitly limit the colchicine used, and it can be any of the commercially available colchicines. In some specific embodiments, the colchicine used is manufactured by Biosan Tech, with production batch number J1240801 and specification of 1 mg / mL.

[0109] Therefore, the application of the above-described embodiments of this application can bring the following technical advantages:

[0110] 1. Targeted solution to the problem of amniotic fluid cells not adhering to the wall due to contamination: This invention takes advantage of the brief time difference (about 40 hours after inoculation) when contaminants such as maternal blood cells have not adhered or are not firmly attached to the wall while fetal cells have initially adhered. The former is precisely removed by physical rinsing, while the latter is preserved.

[0111] 2. Significantly improves clone formation efficiency: Physically removes impurity cells from the slide surface, providing a clean attachment substrate for subsequent fetal cell division and expansion.

[0112] 3. Streamline processes and improve efficiency:

[0113] (1) No complicated pretreatment required: Unlike some methods that require additional steps such as gradient centrifugation and red blood cell lysis, this method only adds a gentle liquid addition and rinsing step within the in situ culture framework.

[0114] (2) Avoid unnecessary passage: Traditional methods often require passage amplification due to the small number of clones. This method increases the initial number of clones, eliminates the passage operation for amplification, shortens the total culture time, and reduces the risks of cell damage, microbial contamination and operation costs caused by passage.

[0115] 4. Improved downstream testing quality: The obtained fetal cell population has higher purity, effectively avoiding contamination by maternal DNA. This is crucial for chromosome karyotype analysis, especially for high-sensitivity molecular detection technologies such as SNP-array, CNV-seq, and NIPS, effectively preventing signal contamination, abnormal typing, false negatives, or false positives caused by sample contamination, ensuring the accuracy and reliability of the test.

[0116] 5. Simple operation and easy standardization: The method and steps are clear, and the key operations (rinsing timing and intensity) are easy to train and standardize, making it suitable for large-scale application in clinical laboratory testing.

[0117] 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 variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should 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 modified in situ culture method for removing maternal blood and impurity cell contamination from amniotic fluid, characterized in that, The method includes the following steps: S1: Sample processing and inoculation: After centrifuging the amniotic fluid sample, discard the supernatant and retain the cell pellet; resuspend the cell pellet using amniotic fluid culture medium to prepare a cell suspension, and inoculate the cell suspension into a culture container containing a growth surface; S2: Initial adherence culture: The culture container after inoculation is placed in static culture at 37°C and 5% CO2 to allow the fetal cells in the amniotic fluid to initially adhere to the growth surface. S3: Key flushing and adding of culture medium: After the initial adherence culture is completed, use a sterile pipette to draw fresh amniotic fluid culture medium and drop the culture medium vertically and stably onto the growth surface from a height of 3-5 cm directly above the sample well of the culture container. Use the physical impact force of the liquid flow to flush the growth surface to remove maternal blood cells that have not adhered or are not firmly attached, as well as impurity cells. S4: Medium change and passage: After continued culture, when more than 10 cell clones with a diameter greater than 2 mm appear on the growth surface, perform medium change or passage. S5: Cell Harvesting: When the cell clones grow to meet the harvesting criteria, pure fetal amniotic fluid cells are harvested.

2. The method according to claim 1, characterized in that, In step S1, the centrifugation conditions are: room temperature, 1500 rpm, centrifugation for 10 minutes.

3. The method according to claim 1, characterized in that, In step S1, the volume of the inoculated cell suspension is 0.5-1 mL.

4. The method according to claim 1, characterized in that, In step S2, the initial adherent culture time is 40±n (n takes the value of 1-3) hours.

5. The method according to claim 1, characterized in that, In step S3, the critical rinsing and liquid addition operation is performed 40 hours after the initial adherence culture, when it is confirmed under a microscope that a few fetal cells have adhered to the culture vessel.

6. The method according to claim 1, characterized in that, In step S3, the fresh amniotic fluid culture medium is preheated to 37°C before use, and the volume of the fresh amniotic fluid culture medium added is 3±n (n takes the value of 1-2) mL.

7. The method according to claim 1, characterized in that, In step S3, after completing the key rinsing and liquid addition, the culture container is returned to 37°C and 5% CO2 conditions for continued culture for 5±n (n takes the value of 1-3) days.

8. The method according to claim 1, characterized in that, In step S4, the liquid replacement operation specifically involves: after aspirating the old culture medium, moving the pipette tip to one end of the growth surface, and slowly and evenly rinsing the entire growth surface with fresh culture medium in a horizontal direction. This rinsing action is repeated twice.

9. The method according to claim 1, characterized in that, In step S5, the harvesting criteria are: at least 15 cell-rich medium-to-large clones on the growth surface, of which clones with a diameter ≥ 6 mm are large clones, and clones with a diameter of 2-6 mm are medium clones; the specific cell harvesting method is as follows: (1) Colchicine treatment: Add colchicine solution to the culture container to make the final concentration about 100 µg / mL, and act on it for 30 minutes at 37℃ and 5% CO2. (2) Hypotonic treatment: After aspirating the culture medium, add a mixture of 0.4% sodium citrate and 0.4% potassium chloride preheated to 37°C and let it stand at 37°C for 30 minutes; (3) Fixation and slide preparation: The slides were fixed three times in sequence with glacial acetic acid pre-fixation and methanol:glacial acetic acid fixation solution with a volume ratio of 3:

1. Then the slides were dried in an environment of 25°C and 50% humidity and baked at 80°C for 3.5 hours. (4) Banding: G-banded chromosome slides were prepared by digestion with trypsin and Giemsa staining.

10. Pure fetal amniotic fluid cells obtained by the method according to any one of claims 1-9 are used for chromosome preparation, karyotype analysis, or digestion followed by collection of precipitates for molecular detection.

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