Preparation method of whole tissue single-cell suspension of human placenta villus tissue

Abstract

The invention relates to the technical field of cell extraction, in particular to a preparation method of a whole tissue single-cell suspension of human placenta villus tissue. The preparation method of the human placenta villus tissue single-cell suspension has the advantages of being small in digestive enzyme dosage, short in digestion time and high in cell activity, the prepared single-cell suspension is high in total cell concentration, the cell activity is 90% or above, the caking rate is low, the placenta villus tissue whole-tissue single-cell suspension can be rapidly prepared in a large scale, operation is easy, and the preparation method is suitable for industrial production. The method is simple in operation and low in reagent cost, can effectively retain the activity of whole tissue cells and the integrity of cell membranes, overcomes the defects of complicated operation, low cell activity and the like in the existing method, and has better application value in the fields of placenta villus tissue cell culture, organoid culture, flow cytometry, single cell sequencing and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of cell extraction, and particularly to a method for preparing a whole-tissue single-cell suspension of human placental villus tissue. Background Art

[0002] The placenta, as the main body of the maternal-fetal interface, is an important organ for ensuring the normal growth and development of the fetus during pregnancy. A normally developed placenta has functions such as regulating maternal-fetal nutrient exchange, immune regulation, secreting hormones and growth factors, which are of great significance for the health of both the mother and the fetus. Poor development of the placenta is related to the incidence of diseases such as preeclampsia and gestational diabetes. Placenta villi tissue, which accounts for the main part of the placenta and develops from trophoblast cells invading the uterine wall in the early pregnancy, is an important site for maternal-fetal material exchange and immune regulation.

[0003] The single-cell dissociation technology of placenta villi tissue can provide reliable samples for subsequent single-cell transcriptome sequencing, flow cytometry detection, organoid culture and other experiments, which helps to detect the differences in cell composition, cell function, gene expression, etc. of placenta villi tissue related to placenta-related diseases, so as to reveal the disease occurrence mechanism, bring more accurate and effective disease diagnosis and treatment methods for patients, and promote the development of prevention and treatment means for related obstetric and gynecological diseases.

[0004] However, there are huge differences in cell composition, matrix composition, cell connection mode, cell sensitivity, etc. between different species and different tissues. The cell dissociation methods of experimental animals and human tissues, as well as different human tissues, cannot be directly migrated and applied, and the corresponding cell dissociation methods need to be optimized according to the species and tissues. Placenta villi tissue has the characteristics of complex structure, numerous cell types, closely connected matrix cells, soft tissue, etc., and is rich in blood supply, and is sensitive to environmental oxygen content, pH, mechanical pressure and digestive enzymes. Existing methods for single-cell isolation of human placenta villi tissue have defects such as long digestion time, low cell viability, cell aggregation, and low integrity of cell membranes and surface receptors. It is often necessary to remove dead cells to improve the proportion of live cells and improve the sample quality, which increases the experimental steps and may affect the success rate and experimental results of subsequent single-cell sequencing and flow cytometry detection. Therefore, the preparation of a whole-tissue single-cell suspension of human placenta villi tissue is a technical difficulty in related research.

[0005] Currently, there are certain deficiencies in the dissociation techniques of human placental villus tissues reported in the literature. For example, there are reports on the application of the Umbilical Cord Dissociation Kit tissue dissociation kit produced by Miltenyi Biotec GmbH in human placental tissues. However, it is expensive and often needs to be used in combination with the same brand's Gentle MACS Dissociator separator and its consumables. The cost of reagents and equipment consumables and the difficulty of obtaining them limit the relevant applications. The Accutase cell digestive solution produced by companies such as Sigma, BD, and Merck Millipore has a relatively low cost, but its dissociation effect is unstable. In addition, there are literature reports that when Accutase is applied to dissociate placental basal plates and villus tissues, the cell viability is only 51.1% before the step of removing dead cells. [1] ( Figure 1 ) For the dissociation reagent using 2.5% Trypsin, 300 U / ml collagenase, 200 µg / ml DNase and PBS as the buffer, the actually detected placental cell viability is about 47%. [2] ( Figure 2 ) Most other literature uses additional dead cell removal steps or purification steps to improve the cell viability in the prepared suspension. [3-4] In addition, US20080064098A1 uses mechanical + enzymatic method + gradient centrifugation to enrich immune cells. The immune cell viability before freezing and thawing is 81.66% (0.81660). The immune cell viability can only reach 98% after flow cytometry screening. Not only does it only screen out about 5 - 10% of the immune cells in the placenta without detecting the viability of all tissue cells after dissociation, but it also has the disadvantages of cumbersome combined use of multiple methods, high prices of instruments and reagents, etc.; in US8591883B2, the average cell viability of most digested tissues (amnion, chorion or amnio-chorionic plate) is only about 70%, and the detection method is trypan blue staining without removing the interference of red blood cell activity, so the detection result may be on the high side. In addition, patents such as WO2010026574A2, US20060030039A1, US20220160787A1, US9096827B2, etc. are only used for cell culture purposes after dissociating single cells. Therefore, there are no requirements for the cell viability of the single cell suspension prepared before dissociation for culture and amplification, and no relevant data are provided; patents such as CN108148806A, CN102703380A, CN108441474B, CN111500529A, CN116731958A, CN101326280A, etc. only involve the rapid separation of placental stem cells, adherent cells, hematopoietic stem cells or decidual stem cells and the subsequent culture of cells or exosomes. The tissues used are different from placental villus tissues, and there are also no requirements for the cell viability after initial enzymatic digestion, nor are relevant data provided.

[0006] In summary, there is still a need to develop an efficient, low-cost, and highly active method for preparing single-cell suspensions from the whole tissue of human placental villi to promote subsequent research in related fields. Summary of the Invention

[0007] The present invention provides a method for preparing a single-cell suspension from the whole tissue of human placental villi, effectively improving the defects such as cumbersome operation and low cell activity in the existing methods.

[0008] To achieve the above object, in a first aspect, the present invention provides a method for preparing a single-cell suspension from human placental villi tissue, the method comprising: enzymatically digesting the placental villi tissue with a dissociation reagent, and then terminating the enzymatic digestion with a dissociation termination solution; Wherein, the dissociation reagent comprises a dissociation enzyme, a buffer, and a solvent. The dissociation enzyme comprises collagenase I, dispase II, and DNase I, and the final concentrations are as follows: collagenase I 0.05 - 2 U / ml, dispase II 0.2 - 20 U / ml, DNase I 5 - 50 U / ml. The solvent is HBSS buffer containing calcium and magnesium.

[0009] Wherein, the dissociation termination solution contains HBSS buffer without calcium and magnesium.

[0010] In the above dissociation reagent, calculated by mass concentration, the final concentrations of collagenase I and dispase II are 0.04 - 16 μg / ml and 0.4 - 40 mg / ml respectively.

[0011] Preferably, the final concentrations of the enzymes in the dissociation reagent are: collagenase I 0.1 - 0.5 U / ml, dispase II 0.6 - 3 U / ml, DNase I 7.5 - 50 U / ml.

[0012] Preferably, the buffer comprises HEPES with a final concentration of 5 - 50 mM.

[0013] Preferably, in the dissociation reagent, the final concentration of HEPES is 10 - 50 mM.

[0014] Preferably, the dissociation reagent further comprises pyruvic acid or its salt with a final concentration of 0.5 - 5 mM. The final concentration of sodium pyruvate is more preferably 1 - 5 mM.

[0015] Preferably, the dissociation reagent uses HBSS buffer containing calcium and magnesium as the solvent, and comprises the following components: collagenase I 0.1 - 0.5 U / ml, dispase II 0.6 - 3 U / ml, DNase I 7.5 - 50 U / ml, HEPES 5 - 50 mM, sodium pyruvate 0.5 - 5 mM.

[0016] Preferably, the dissociation termination solution further contains 2-10% FBS.

[0017] In some embodiments of the present invention, the dissociation termination solution is calcium- and magnesium-free HBSS buffer supplemented with 2-10% FBS.

[0018] Based on the above reagent optimization, the temperature, shaking speed, and digestion time of enzymatic digestion were further optimized. In the above-described preparation method, the enzymatic digestion is carried out under heating and shaking conditions of 20-37 °C and 30-200 rpm for 5-30 min.

[0019] Preferably, the enzymatic digestion conditions are: enzymatic digestion for 10-30 min under the conditions of 35-37 °C and 60-100 rpm.

[0020] In the above preparation method, after terminating the enzymatic digestion, it further includes the steps of filtering through a cell sieve and removing red blood cells.

[0021] In the above preparation method, before enzymatic digestion, it further includes the steps of sampling, preserving, and trimming the placental villous tissue; during the sampling, preserving, and trimming process, the buffer used is calcium- and magnesium-containing HBSS buffer.

[0022] In a second aspect, the present invention provides a reagent set or kit for preparing a single-cell suspension of human placental villous tissue, the reagent set or kit includes a dissociation reagent and a dissociation termination solution; the dissociation reagent and the dissociation termination solution are as described in the first aspect above.

[0023] The above reagent set for preparing a single-cell suspension of human placental villous tissue can be prepared as a complete set of reagents or a kit for preparing a whole-tissue single-cell suspension of human placental villous tissue.

[0024] In a third aspect, the present invention provides the application of the above-described reagent set or kit for preparing a single-cell suspension of human placental villous tissue in the preparation of a single-cell suspension of human placental villous tissue.

[0025] Specifically, the application includes the following steps: (1) Obtain a placental sample, place it in calcium- and magnesium-containing HBSS buffer for preservation and transportation; after collecting the placental villous tissue, wash and trim it; (2) Use the dissociation reagent to enzymatically digest the placental villous tissue in step (1); (3) Use the dissociation termination solution to terminate the enzymatic digestion; after filtering through a cell sieve, wash the cell sieve with the dissociation termination solution, combine the filtrates, centrifuge to remove the supernatant, resuspend the cell pellet with red blood cell lysate to remove red blood cells, add calcium- and magnesium-containing HBSS buffer to terminate the lysis, and centrifuge and resuspend to prepare a single-cell suspension.

[0026] Preferably, in the above step (1), the method for collecting placental villus tissue includes: cutting a tissue block of about (1-3) cm × (5-7) cm from the edge of the fresh placenta towards the center of the placenta with scissors and quickly placing it in HBSS buffer containing calcium and magnesium. The method for trimming the tissue is: taking out the tissue block and placing it in HBSS buffer containing calcium and magnesium, removing about 0.5 cm deep amnion on the fetal side and decidua basalis on the maternal side, taking about 4-6 g of placental villus tissue in the middle of the placenta, and cutting it into small pieces of about 0.3 mm 3 with scissors; rinsing 2-3 times with HBSS buffer containing calcium and magnesium. If there is obvious blood, repeat the washing and remove obvious blood clots; transferring the tissue to HBSS buffer containing calcium and magnesium and cutting it into small pieces of about 0.1 mm 3 with scissors, allowing it to stand and then discarding the supernatant. During the sampling, preservation, and trimming processes, the buffer used is HBSS buffer containing calcium and magnesium.

[0027] In the above step (2), a dissociation reagent is added to the placental villus tissue obtained by trimming in step (1) and enzymatic digestion is carried out. The dosage of the dissociation reagent is 1-8 ml / g of tissue (preferably 2-3 ml / g of tissue).

[0028] In the above step (3), the dosage of the dissociation termination solution added is 1-5 times the volume of the dissociation reagent.

[0029] In the above step (3), the pore size of the cell sieve used for filtration is 40-100 μm.

[0030] In the above step (3), the centrifugation conditions are 200-400×g, 3-10 min.

[0031] In the above step (3), the conditions for red blood cell lysis are: at room temperature, 5-10 min. After red blood cell lysis treatment, centrifugation is carried out, and the centrifugation conditions are 200-400×g, at room temperature, 3-10 min. After centrifugation, the cells are resuspended with HBSS buffer containing calcium and magnesium.

[0032] Compared with the prior art, the advantages of the present invention are: 1. Optimize the selection and dosage of dissociating enzymes in a one-step method. Using dispase II as the main working enzyme, reduce the dissociation time and improve cell viability under the same yield: The present invention finds that dispase II is relatively mild and has the least impact on the activity of human placental villous tissue cells and the integrity of cell membranes. Therefore, it has the largest dosage in the formula. Hyaluronidase, type IV collagenase, and the combination of type IV collagenase and type I collagenase, which are commonly used in the preparation of single-cell suspensions of other tissues, cause greater damage to the activity of cells in human placental villous tissue, so they are not selected. Type I collagenase can degrade collagen fibers to promote cell dissociation and significantly reduce damage to placental villous tissue compared to type IV collagenase. DNase I can degrade the released DNA to prevent cell aggregation. The present invention also finds that the combination of dispase II and type I collagenase with dispase II as the main working enzyme can improve the dissociation efficiency and protect cell viability. In addition, the present invention finds that under the preferred conditions, the activity of placental villous tissue still gradually decreases with the length of the dissociation time. Therefore, the dissociation time is optimized and simplified to a one-step dissociation method, removing the mechanical pressure caused by multiple steps of operation and the influence of prolonged dissociation time, and improving work efficiency.

[0033] 2. The present invention also designs and verifies the beneficial effects of alternating the use of two types of HBSS on reducing the enzyme activity dosage and precisely controlling the dissociation process, reducing the dissociating enzyme dosage by 2 - 3 orders of magnitude compared with similar reports: The HBSS buffer solution containing calcium and magnesium, compared with commonly used phosphate buffer solutions such as PBS, utilizes the characteristics of calcium and magnesium ions to activate dispase II and type I collagenase, reducing the working concentration of the required type I collagenase from the mg / ml level to the μg / ml level. It is reduced from the commonly reported 125U - 300U / ml to 0.05 - 2 U / ml, further improving the dissociation efficiency and protecting cell viability. The dosage of the dissociating enzyme used is much lower than the conventional dosage reported in the literature. At the same time, the dissociation termination solution correspondingly uses an HBSS buffer solution without calcium and magnesium. Combining with a lower enzyme activity dosage can effectively terminate the dissociation and control the reaction progress. In addition, FBS in the dissociation termination solution can cooperate to terminate the dissociation and prevent cell aggregation.

[0034] 3. Considering the characteristics of highly metabolic cells, use HBSS and add a buffer: Glucose contained in HBSS and additionally added sodium pyruvate can further provide energy for cells. The added HEPES can also buffer the environmental acidification caused by the respiratory process of highly metabolic cells in human placental villous tissue during storage and incubation, better preserving the viability of highly metabolic cells. Using sterile HBSS for preservation during transportation can also reduce the geographical restrictions of the sampling location.

[0035] Therefore, by exploring the sensitivity of human placental villous tissue to the composition of digestive enzymes, enzymatic hydrolysis temperature, and time, and combining with the characteristics of cell metabolism, the present invention optimizes the optimal concentrations of dispase II, collagenase I, and DNase I, and optimizes parameters such as dissociation and termination dissociation reagents and dissociation time. Compared with commercially available kits or similar reports, it has the advantages of cell viability of more than 90% in whole tissue, simple operation, low dosage of digestive enzymes, low preparation cost, and high preparation efficiency. It is applicable to cell culture, organoid culture, flow cytometry detection, single-cell sequencing, etc. after rapid large-scale preparation, and helps to improve the success rate of subsequent detection, providing effective method support for further exploring the pathogenesis and treatment strategies of related diseases. Description of the Drawings

[0036] Figure 1 It is a result diagram of counting cell viability after preparing a single-cell suspension of Document 1 in the background art of the present invention.

[0037] Figure 2 It is the result of AO / PI staining after preparing a single-cell suspension of Document 2 in the background art of the present invention.

[0038] Figure 3 It is a flow chart of the method for preparing a single-cell suspension of human placental villous tissue of the present invention.

[0039] Figure 4 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Example 1 of the present invention.

[0040] Figure 5 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 1 of the present invention.

[0041] Figure 6 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Example 2 of the present invention.

[0042] Figure 7 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 2 of the present invention.

[0043] Figure 8 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Example 3 of the present invention.

[0044] Figure 9 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 3 of the present invention.

[0045] Figure 10 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 4 of the present invention.

[0046] Figure 11It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 5 of the present invention.

[0047] Figure 12 It is the result of AO / PI staining of the single-cell suspension prepared by the method of Comparative Example 6 of the present invention. Detailed implementation manners

[0048] The technical content, implementation steps, and technical effects of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. It is intended to explain the present invention and should not be construed as a limitation to the present invention. The present invention is not limited to the following specific embodiments.

[0049] In the following examples and comparative examples, the DNase I used was purchased from Biofroxx, product number 1121MG010 (750U / mg); the collagenase I used was purchased from Sigma, product number C0130-100MG (>125CDU / mg); the dispase II used was purchased from Sigma, product number D4693-1G (>0.5U / mg); the sodium pyruvate used was purchased from Sigma, product number P2256-5G; the HEPES solution used was purchased from Macklin, product number H917699-250ml; the HBSS buffer used was purchased from Senrui Biotech, product numbers CR14175-1 (calcium- and magnesium-free) and CR-14025 (calcium- and magnesium-containing); the Accutase used was purchased from Invitrogen, product number 00-4555-56; the FBS used was purchased from Gibco, product number 10099141c; the red blood cell lysate used was purchased from Beyotime, product number C3702. The AOPI used was purchased from Nexcelom, product number CS2-0106-5ML. The PBS used was purchased from Biosharp, product number BL302A. The collagenase IV used was purchased from Thermo Fisher, product number 17104019; the hyaluronidase used was purchased from Aladdin, product number H304866-25mg.

[0050] Example 1 This example provides a reagent set for the preparation of a single-cell suspension of human placental villus tissue, which includes a dissociation reagent and a dissociation termination solution; wherein, the dissociation reagent includes components with the following final concentrations: dispase II 0.8U / ml, collagenase I 0.3U / ml, DNase I 37.5U / ml, sodium pyruvate 1mM, HEPES 10mM, and the solvent is a calcium- and magnesium-containing HBSS buffer; the dissociation termination solution includes the following components: 2% FBS, and the solvent is a calcium- and magnesium-free HBSS buffer.

[0051] This example also provides a method for preparing a single-cell suspension of human placental villus tissue based on the above reagent set for the preparation of a single-cell suspension of human placental villus tissue, specifically including the following steps (the process schematic diagram is asFigure 3 as shown in (1)Tissue sampling and transportation: Check the appearance of the placenta, place the maternal surface upwards, gently remove the blood clots on the surface and the fetal membranes covering the placenta surface. Use tissue forceps to pick up the fetal membranes at the edge of the placenta, avoiding directly picking up the placental tissue. Use surgical scissors to cut a tissue of about 2 cm × 6 cm from the edge of the placenta towards the center of the placenta. Rinse the sample with sterile HBSS buffer containing calcium and magnesium, and transport it in HBSS buffer containing calcium and magnesium.

[0052] (2)Tissue cleaning and shearing: After sending to the laboratory, place it in a 100 mm petri dish containing HBSS buffer with calcium and magnesium for trimming. Remove about 0.5 cm thick amnion on the fetal surface and decidua basalis on the maternal surface. Take 5 g of placental villous tissue in the middle of the placenta and cut it into small pieces of about 0.3 mm 3 in size, rinse it 2 - 3 times with HBSS buffer containing calcium and magnesium. If there is obvious blood, repeat the washing and carefully remove the obvious blood clots with forceps. Transfer the tissue to a 50 ml centrifuge tube containing 30 ml of HBSS buffer with calcium and magnesium, and cut it into small pieces of about 0.1 mm 3 in size. Let it stand and discard the supernatant. During the trimming process, pay attention not to squeeze the tissue block forcefully.

[0053] (3)Enzymatic digestion treatment: Add 10 ml of dissociation reagent at 37 °C to the shredded tissue blocks obtained in step (2), and place it on a heating shaker for enzymatic digestion. The enzymatic digestion conditions are: 37 °C, 90 rpm, 10 min.

[0054] (4)Terminate dissociation and filter: Add 30 ml of dissociation termination solution at 4 °C to terminate dissociation, and filter it through a 70 μm cell strainer into a 50 ml centrifuge tube. Wash the strainer with 10 ml of dissociation termination solution, combine the filtrates, and centrifuge (290 × g, room temperature, 5 min) to remove the supernatant.

[0055] (5)Lyse red blood cells, resuspend, and quality inspection: Add 6 ml of red blood cell lysate and lyse at room temperature for 5 min. Add an equal volume of HBSS buffer with calcium and magnesium to terminate red blood cell lysis. After centrifugation (290 × g, room temperature, 5 min), add 5 ml of HBSS buffer with calcium and magnesium to resuspend, obtaining a single - cell suspension of placental villous tissue.

[0056] Gently pipette the single - cell suspension of placental villous tissue evenly, then take 10 μL of the suspension and add an equal volume of AOPI, and count using a cell counter (CountStar). The results are shown in Figure 4 , and the results show that the single - cell viability rate of the whole placental villous tissue can reach 98.95%, the total cell concentration is 9.96 × 10 5 cells / ml, the viable cell concentration is 9.86 × 10 5 cells / ml, and the aggregation rate is 2.04%.

[0057] Comparative Example 1 This comparative example provides a method for preparing a single-cell suspension of placental villus tissue based on the commercially available Invitrogen Accutase cell digestive solution. The difference from the single-cell suspension preparation method of Example 1 is only that: the dissociation reagent used in step (3) of the single-cell suspension preparation method of Example 1 is replaced with Invitrogen Accutase cell digestive solution.

[0058] The results are shown in Figure 5 , and the results show that the overall single-cell viability of the obtained placental villus tissue is 88.96%, the total cell concentration is 7.47×10 5 cells / ml, the viable cell concentration is 6.65×10 5 cells / ml, and the aggregation rate is 4.33%. The results suggest that when using Accutase, with the same pre-preparation steps and the same enzymatic digestion time (10 min), the cell viability is relatively low and the total number of cells obtained is small.

[0059] Example 2 This example provides a reagent set for preparing a single-cell suspension of human placental villus tissue, which includes a dissociation reagent and a dissociation termination solution; wherein, the dissociation reagent includes components with the following final concentrations: dispase II 0.6 U / ml, collagenase I 0.1 U / ml, DNase I 7.5 U / ml, sodium pyruvate 5 mM, HEPES 50 mM, and the solvent is HBSS buffer containing calcium and magnesium; the dissociation termination solution includes the following components: 2% FBS, and the solvent is HBSS buffer without calcium and magnesium.

[0060] This example also provides a method for preparing a single-cell suspension of human placental villus tissue based on the above reagent set for preparing a single-cell suspension of human placental villus tissue. The specific steps are different from those of Example 1 in that the enzymatic digestion conditions are: 37 °C, 60 rpm, 30 min.

[0061] The results are shown in Figure 6 , and the results show that the overall single-cell viability of the placental villus tissue is 93.84%, the total cell concentration is 8.52×10 5 cells / ml, the viable cell concentration is 8.00×10 5 cells / ml, and the aggregation rate is 3.15%.

[0062] Comparative Example 2 This comparative example provides a method for preparing a single-cell suspension of placental villus tissue based on the commercially available Invitrogen Accutase cell digestive solution. The difference from the single-cell suspension preparation method of Example 2 is only that: the dissociation reagent used in step (3) of the single-cell suspension preparation method of Example 2 is replaced with Invitrogen Accutase cell digestive solution.

[0063] The results are shown in Figure 7 , and the results show that the overall single-cell viability of the obtained placental villus tissue is 63.26%, the total cell concentration is 4.70×10 5 cells / ml, the viable cell concentration is 2.97×10 5 cells / ml, and the aggregation rate is 1.79%. The results suggest that when using Accutase, with the same pre-preparation steps and the same extended enzymatic digestion time up to 30 min, the cell viability decreases significantly, and the obtained cell quantity fluctuates greatly, and is much less than the preparation method of the embodiment of the present invention.

[0064] Example 3 This example provides a reagent set for preparing a single-cell suspension of human placental villus tissue, which includes a dissociation reagent and a dissociation termination solution; wherein, the dissociation reagent includes components with the following final concentrations: dispase II 3.0 U / ml, collagenase I 0.5 U / ml, DNase I 50 U / ml, sodium pyruvate 1 mM, HEPES 10 mM, and the solvent is HBSS buffer containing calcium and magnesium. The dissociation termination solution includes the following components: 2% FBS, and the solvent is HBSS buffer without calcium and magnesium.

[0065] This example also provides a method for preparing a single-cell suspension of human placental villus tissue based on the above-mentioned reagent set for preparing a single-cell suspension of human placental villus tissue. The specific steps are different from those of Example 1 in that the enzymatic digestion time is 15 min.

[0066] The results are shown in Figure 8 , and the results show that the overall single-cell viability of the obtained placental villus tissue is 97.74%, the total cell concentration is 8.63×10 5 cells / ml, the viable cell concentration is 8.43×10 5 cells / ml, and the aggregation rate is 3.60%. The results suggest that using this preparation protocol has a slight impact on cell viability on the premise of increasing the dosage of collagenase I and DNase and the enzymatic digestion time.

[0067] Comparative Example 3 This comparative example provides a method for preparing a single-cell suspension of placental villus tissue based on a PBS cell digestive solution, and the difference from the single-cell suspension preparation method of Example 3 is only that: the dissociation reagent used in step (3) is replaced with the following formula: 0.8 U / ml dispase II, 0.2 U / ml collagenase I, 7.5 U / ml DNase I, 1 mM sodium pyruvate, 10 mM HEPES, and the solvent is PBS buffer solution.

[0068] The results are shown in Figure 9 , and the results show that the overall single-cell viability of the obtained placental villus tissue is 96.40%, the total cell concentration is 1.67×10 5 cells / ml, the viable cell concentration is 1.61×10 5 cells / ml, and the aggregation rate is 2.33%. The results suggest that using PBS instead of HBSS buffer containing calcium and magnesium, the cell viability slightly decreases under the same dissociation time, and the number of obtained cells is much smaller than that of the preparation method of the embodiment of the present invention.

[0069] Comparative Example 4 This comparative example provides a method for preparing a single-cell suspension of placental villus tissue based on a PBS cell digestive solution, and the difference from the single-cell suspension preparation method of Example 3 is only that: the dissociation reagent used in step (3) in the single-cell suspension preparation method of Example 3 is replaced with the following formula: 320 U / ml collagenase IV, 50 U / ml DNase I, and the solvent is PBS buffer solution.

[0070] The results are shown in Figure 10 , and the results show that the overall single-cell viability of the obtained placental villus tissue is 1.30%, the total cell concentration is 6.93×10 5 / ml, the viable cell concentration is 9.00×10 3 / ml, and the aggregation rate is 25.58%. The results suggest that collagenase IV has a great impact on the activity of human placental villus whole tissue cells under the working concentration, buffer solution and dissociation time reported for other species or tissues.

[0071] Comparative Example 5 This comparative example provides a method for preparing a single-cell suspension of placental villus tissue based on an HBSS cell digestive solution, and the difference from the single-cell suspension preparation method of Example 3 is only that the dissociation reagent used in step (3) in the single-cell suspension preparation method of Example 3 is replaced with the following formula: 0.2 U / ml collagenase IV, 0.2 U / ml collagenase I, 50 U / ml DNase I, and the solvent is HBSS buffer containing calcium and magnesium.

[0072] The results are shown in Figure 11, The results showed that the single-cell viability of the obtained placental villous tissue was 4.48%, the total cell concentration was 1.05×10 7 cells / ml, the viable cell concentration was 4.73×10 5 cells / ml, and the aggregation rate was 19.74%. The results indicated that adding a low concentration of collagenase IV in the embodiment of the present invention, under the same previous preparation steps and the same enzymatic digestion time, although slightly more cell numbers were obtained than those in the preparation method of the example, the cell viability was significantly reduced. Together with Comparative Example 4, it demonstrated that collagenase IV significantly reduced the cell viability of human placental villous tissue.

[0073] Comparative Example 6 This comparative example provides a method for preparing a single-cell suspension of placental villous tissue based on HBSS cell digestive solution. The difference between its single-cell suspension preparation method and that of Example 3 is only that the dissociation reagent used in step (3) of Example 3 is replaced with the following formula: 10 U / ml hyaluronidase, 0.2 U / ml dispase II, 0.125 U / ml collagenase I, 50 U / ml DNase I, and the solvent is HBSS buffer containing calcium and magnesium.

[0074] The results are shown in Figure 12 , The results showed that the single-cell viability of the obtained placental villous tissue was 55.73%, the total cell concentration was 7.46×10 5 cells / ml, the viable cell concentration was 4.16×10 5 cells / ml, and the aggregation rate was 29.46%. The results indicated that adding a low concentration of hyaluronidase reported in other species or tissues in the embodiment of the present invention, under the same previous preparation steps and the same enzymatic digestion time, the obtained cell numbers were significantly reduced and the cell viability was significantly reduced. It was shown that the effect of combining collagenase I and hyaluronidase was inferior to that of the embodiment of the present invention in the single-cell preparation of human placental villous tissue.

[0075] Table 1 statistically shows the effects of Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, Example 3 and Comparative Examples 3, 4, 5, 6 on the cell viability and cell numbers of the whole tissue under different final concentrations of dissociation enzymes and dissociation time conditions.

[0076] Table 1: Single-cell suspensions of human placental villous tissue obtained with different combinations of dissociation solutions and enzymatic digestion times

[0077] In summary, the method for preparing a single-cell suspension of human placental villous tissue provided in the embodiments of the present invention has the characteristics of a large total number of cells, high cell viability, and low aggregation rate in the preparation of the single-cell suspension, and has obvious advantages compared with the methods in the comparative examples.

[0078] References: [1] Afshar Y, Yin O, Jeong A, et al. Placenta accreta spectrum disorder at single-cell resolution: a loss of boundary limits in the decidua and endothelium[J]. American journal of obstetrics and gynecology , 2024, 230(4): 443. e1-443. e18. [2] Garcia-Flores, V., Xu, Y., Pusod, E.et al. Preparation of single-cell suspensions from the human placenta[J]. Nature protocols , 2023, 18(3):732-754. [3] Admati I, Skarbianskis N, Hochgerner H, et al. Two distinct molecular faces of preeclampsia revealed by single-cell transcriptomics[J]. Med , 2023, 4(10): 687-709. e7. [4] Vento-Tormo R, Efremova M, Botting R A, et al. Single-cell reconstruction of the early maternal–fetal interface in humans[J]. Nature ,2018, 563(7731): 347-353. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a single cell suspension of human placental villus tissue, characterized in that: The method comprises: using a dissociation reagent to enzymatically hydrolyze the placental villus tissue, and then using a dissociation stop solution to stop the enzymatic hydrolysis; The dissociation reagent includes a dissociation enzyme, a buffer and a solvent; wherein the dissociation enzyme includes collagenase I, dispase II and DNase I, and the final concentrations thereof are as follows: collagenase I 0.05-2U / ml, dispase II 0.2-20U / ml, DNase I 5-50U / ml; the solvent is a calcium-magnesium-containing HBSS buffer; The dissociation termination solution comprises HBSS buffer without calcium and magnesium.

2. The preparation method according to claim 1, characterized in that: The buffer contained HEPES at a final concentration of 5-50 mM.

3. The preparation method according to claim 1 or 2, characterized in that: The dissociation reagent further comprises pyruvic acid or a salt thereof at a final concentration of 0.5-5 mM.

4. The preparation method according to any one of claims 1 to 3, characterized in that The dissociation stop solution also contains 2-10% FBS.

5. The preparation method according to any one of claims 1 to 4, characterized in that The enzymolysis is carried out at 20-37° C. and 30-200 rpm heating and shaking conditions for 5-30 min.

6. The preparation method according to any one of claims 1 to 5, characterized in that After the enzymatic hydrolysis is terminated, the steps of filtering through a cell mesh and removing red blood cells are also included.

7. The preparation method according to any one of claims 1 to 5, characterized in that Before enzymatic hydrolysis, the process also includes the steps of sampling, preserving and trimming the placental villus tissue; During the sampling, storage and trimming process, the buffer used is HBSS buffer containing calcium and magnesium.

8. A reagent set or kit for preparing a single cell suspension of human placental villus tissue, characterized in that: The reagent set or kit includes a dissociation reagent and a dissociation stop solution; The dissociation reagent includes a dissociation enzyme, a buffer and a solvent; the dissociation enzyme includes collagenase I, dispase II and DNase I, and the final concentrations thereof are as follows: collagenase I 0.05-2U / ml, dispase II 0.2-20U / ml, DNase I 5-50U / ml; the solvent is a calcium-magnesium-containing HBSS buffer; The dissociation termination solution comprises HBSS buffer without calcium and magnesium.

9. The reagent set or kit according to claim 8, characterized in that: The buffer contains HEPES at a final concentration of 5-50 mM; And / or, the dissociation reagent further comprises pyruvic acid or a salt thereof at a final concentration of 0.5-5 mM; And / or, the dissociation stop solution further comprises 2-10% FBS.

10. Use of the reagent set or kit according to any one of claims 8 to 9 in preparing a single cell suspension of human placental villus tissue.

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