Extraction method for lymphangial endothelial cell cyst fluid of lymphangial malformation
By directly collecting and simplifying the cystic fluid from lymphatic malformations, the problems of low cell purity and poor cell viability in traditional methods are solved, providing a high-quality cell model for lymphatic malformation research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEST CHINA HOSPITAL SICHUAN UNIV
- Filing Date
- 2026-01-12
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies for treating lymphatic malformations present challenges in obtaining samples through surgical tissue removal, resulting in low cell purity and poor viability. Furthermore, the procedures are cumbersome, making it difficult to provide high-quality cell models.
By collecting cystic fluid from patients with lymphatic malformations, performing low-speed centrifugation, erythrocyte lysis, and cell culture, combined with immunofluorescence staining for identification, the operation steps are simplified, and cell purity and activity are improved.
This method enables the low-invasive, simple, and efficient acquisition of high-purity lymphatic endothelial cells, making them suitable for basic research and clinical translation, and improving cell culture success rate and purity.
Smart Images
Figure CN122012391A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological cell extraction technology, and in particular to a method for extracting cyst fluid from lymphatic endothelial cells of malformed lymphatic vessels. Background Technology
[0002] Lymphatic malformation (LM) is a vascular disease caused by abnormally developed lymphatic vessels, commonly found in the neck, face, armpits, and mediastinum in children. The cystic cavity often contains a large amount of clear fluid, rich in exfoliated lymphatic endothelial cells, inflammatory cells, and various bioactive substances.
[0003] However, current clinical cytological and molecular studies of LM lesions mostly rely on primary cell isolation and culture from surgically removed tissue. This process is highly invasive to children, difficult to obtain samples, and the surgical samples contain a lot of fibrous tissue and red blood cell contamination, resulting in high purification difficulty, low cell viability, and unstable culture success rate. Summary of the Invention
[0004] The purpose of this invention is to provide a method for extracting cyst fluid from lymphatic endothelial cells in lymphatic vessels with malformations, thereby solving the problems of cumbersome steps, low cell purity, and poor early growth viability in traditional tissue separation methods, and providing a high-quality cell model for basic research and clinical translation related to lymphatic vessels with malformations.
[0005] This invention provides a method for extracting fluid from the endothelial cells of lymphatic vessels with malformations, comprising the following steps:
[0006] S1. Sample collection: Collect 5-10 mL of cystic fluid from patients with large cystic lymphatic malformations and place it in a sterile EP tube or centrifuge tube.
[0007] S2. Initial centrifugation: Use low-speed centrifugation. After centrifugation, discard the supernatant and retain the cell pellet at the bottom.
[0008] S3, Red blood cell lysis: Add an appropriate amount of red blood cell lysis buffer to the cell pellet described in step S2, mix well, and place at 4°C for an appropriate time. After placement, centrifuge, discard the supernatant, wash with PBS, and centrifuge again.
[0009] S4. Cell Culture: Resuspend the cell pellet treated in step S3 in a culture medium containing endothelial growth factor supplement, seed it into 6-well plates or culture flasks, and incubate in an incubator; after 24 hours of culture, change the culture medium and remove non-adherent cells.
[0010] S5 cell identification: Immunofluorescence staining can be performed to identify cells 48-72 hours after cell adhesion.
[0011] Furthermore, in step S1, a sterile syringe is used to collect the cyst fluid.
[0012] Furthermore, the low-speed centrifugation speed in step S2 is 1000 rpm, and the centrifugation time is 10 minutes.
[0013] Furthermore, the appropriate time mentioned in step S3 is 3 to 5 minutes; the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes.
[0014] Furthermore, the conditions for incubation in step S4 are 37°C and 5% CO2.
[0015] Compared with the prior art, the beneficial effects of the present invention are:
[0016] (1) Less invasive: Only a sample of cyst fluid needs to be extracted, without surgical removal of tissue, greatly reducing the risk of injury to patients.
[0017] (2) Simple operation and short time: No collagenase or trypsin digestion is required, and the initial separation of cells can be completed in 30 minutes in a single operation.
[0018] (3) Higher cell purity: The cyst fluid is derived from relatively abundant endothelial cells, with less contamination after erythrocyte lysis, and stable expression of endothelial markers (such as CD31, LYVE-1, D2-40, VEGFR3).
[0019] (4) High culture success rate: The cells have good activity, fast adhesion, and uniform morphology, making them suitable for subsequent immunofluorescence, molecular experiments and drug sensitivity testing.
[0020] (5) High reproducibility and generalizability: It has low requirements for sample size, capsule fluid source and experimental conditions, and is suitable for standardized operation of multi-center samples. Attached Figure Description
[0021] Figure 1 This is a schematic flowchart of a method for extracting endothelial cell cyst fluid from lymphatic vessels with malformations, as disclosed in an embodiment of this application.
[0022] Figure 2 A schematic diagram of microscopic observation on day 3 of primary culture using the LMLEC conventional tissue isolation method;
[0023] Figure 3 A schematic diagram of microscopic observation on day 6 of primary culture using the LMLEC conventional tissue isolation method;
[0024] Figure 4 A schematic diagram of microscopic observation on day 9 of primary culture using the LMLEC conventional tissue isolation method;
[0025] Figure 5 A schematic diagram of microscopic observation on day 3 of primary culture using the LMLEC capsule fluid extraction method;
[0026] Figure 6 A schematic diagram of microscopic observation on day 6 of primary culture using the LMLEC capsule fluid extraction method;
[0027] Figure 7 Immunofluorescence staining of CD31, D2-40, LYVE-1, and VEGFR3 was performed on LMLEC.
[0028] Figure 8 For the detection of positive rate in traditional tissue separation methods using flow cytometry; Figure 8 In Figure A, the CD31 positive rate was detected by flow cytometry using the LMLEC conventional tissue separation method. Figure 8 In the middle section, the positive rate of D2-40 flow cytometry was detected using the LMLEC traditional tissue separation method.
[0029] Figure 9 For the detection of positive rate of LMLEC capsule fluid extraction by flow cytometry; Figure 9 In section A, CD31 positivity was detected by flow cytometry using the LMLEC cyst fluid extraction method. Figure 9 In the study, the positive rate of D2-40 flow cytometry was detected using the LMLEC capsule fluid extraction method.
[0030] Figure 10 Comparison of CD31 flow cytometry positivity rates between traditional tissue separation and cyst fluid extraction methods in LMLEC;
[0031] Figure 11 Comparison of D2-40 flow cytometry positive rates between traditional tissue separation and cyst fluid extraction methods in LMLEC;
[0032] Figure 12 Comparison of cell adhesion rate on day 6 between LMLEC traditional tissue separation method and capsule fluid extraction method. Detailed Implementation
[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0034] The detailed technical solution of the present invention and the corresponding comparative verification experiments will be described in detail below.
[0035] The technical solution of the capsule fluid extraction method of this invention:
[0036] The method for extracting endothelial cell vesicle fluid from abnormal lymphatic vessels provided by this invention specifically includes the following steps:
[0037] S1. Sample collection: Collect 5-10 mL of cystic fluid from patients with large cystic lymphatic malformations using a sterile syringe. Immediately place the collected cystic fluid into a sterile EP tube or centrifuge tube. To ensure cell viability, the sample should be transported to the laboratory as soon as possible (within 2 hours) at 4°C for further processing.
[0038] S2. Initial centrifugation: Place the sterile centrifuge tube containing the cyst fluid in a centrifuge and centrifuge at a low speed of 1000 rpm for 10 minutes. After centrifugation, carefully discard the supernatant and retain the cell pellet at the bottom.
[0039] S3, Red blood cell lysis: Add an appropriate amount of red blood cell lysis buffer to the cell pellet obtained in step S2, gently invert to mix, and incubate at 4°C for 3-5 minutes to fully lyse the mixed red blood cells; then centrifuge again at 1000 rpm for 5 minutes and discard the supernatant containing lysed red blood cells; add PBS buffer to the pellet for washing, and centrifuge again at 1000 rpm for 5 minutes after washing, discard the supernatant, and obtain the purified cell pellet.
[0040] S4. Cell Culture: Resuspend the purified cell pellet from step S3 in complete endothelial cell culture medium containing endothelial growth factor supplement, and gently pipette to mix and prepare a cell suspension; seed the cell suspension into 6-well plates or cell culture flasks and place them in a constant temperature incubator at 37°C and 5% CO2 for primary culture; after 24 hours of culture, replace the medium with fresh medium containing endothelial growth factor supplement, and remove any non-adherent dead cells or impurity cells, and continue culturing to obtain primary lymphatic endothelial cells that adhere to the culture vessel.
[0041] S5 cell identification: After the cells adhere to the wall, the cell phenotype is identified by immunofluorescence staining 48-72 hours later. Optionally, to further improve the accuracy of identification, flow cytometry can be used for quantitative analysis to confirm that the obtained cells are lymphatic endothelial cells.
[0042] Example 1: Comparative verification experiment between the method of the present invention and the traditional method
[0043] To verify the superiority of the capsule fluid extraction method of the present invention, this embodiment uses the traditional tissue separation method as a control and conducts a comprehensive comparative analysis in terms of experimental materials, methods, results and conclusions.
[0044] 1. Experimental Materials
[0045] 1.1 Reagents: PBS buffer (containing 1% penicillin / streptomycin), red blood cell lysis buffer, complete endothelial cell culture medium, 0.25% trypsin-EDTA, collagenase type IV, DNase I, cell cryopreservation solution;
[0046] 1.2 Consumables: Sterile centrifuge tubes (15mL, 50mL), cell culture dishes, cell filters (100μm);
[0047] 1.3 Instruments: centrifuge, clean bench, incubator, microscope, flow cytometer.
[0048] 2. Experimental Methods
[0049] 2.1 Sample pretreatment and primary cell isolation
[0050] This experiment set up two parallel treatments: the experimental group used the capsule fluid extraction method of the present invention (steps are the same as S1~S4 above); the control group used the traditional tissue separation method, the specific steps of which are as follows:
[0051] Control group (traditional tissue separation method): In a clean bench, tissue specimens from patients with macrocystic lymphangioma were transferred to PBS buffer containing 2% penicillin-dextrin antibodies and rinsed repeatedly 3-5 times until the rinsing solution was clear to remove blood and contaminants from the tissue surface. Using sterile surgical scissors and forceps, visible adipose tissue, fibrous capsules, and necrotic parts were carefully removed, preserving the target tissue. The target tissue was then cut into small pieces of 1-2 mm³ and transferred to a solution containing collagenase IV and DNase. In a mixed digestion solution (tissue to digestion solution volume ratio approximately 1:5), centrifuge tubes were placed in a 37°C water bath shaker and gently shaken for 1 hour, during which the digestion status was observed under a microscope periodically. Digestion was immediately stopped when a large number of single cells or small cell clusters were released. An equal volume of serum-containing culture medium was added to neutralize the digestion solution and terminate the digestion reaction. The cell suspension was filtered through a 100μm cell filter to remove undigested large tissue fragments. The filtered cell suspension was collected, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in erythrocyte lysis buffer, and then resuspended in complete endothelial cell culture medium. The cell suspension was seeded into pre-coated culture dishes and incubated in a 37°C, 5% CO2 incubator. After 24 hours of incubation, the culture medium was replaced with fresh medium, and non-adherent cells were removed.
[0052] 2.2 Cell Expansion
[0053] During cell culture in both the experimental and control groups, cell morphology and growth fusion were observed daily under a microscope. When the cell fusion rate reached 80%-90%, the cells were digested and passaged using 0.25% trypsin-EDTA. The cells were then cultured to the 2nd-3rd generation for subsequent cell purity identification.
[0054] 2.3 Cell Identification Methods
[0055] Cell phenotypes were identified using a combination of immunofluorescence staining and flow cytometry. The markers detected included: the pan-endothelial cell positive marker CD31 (PECAM-1), and the lymphatic endothelial cell-specific markers Podoplanin (D2-40), VEGFR-3 (FLT4), and LYVE-1. Immunofluorescence staining was used to observe the protein localization of the markers, while flow cytometry was used to quantitatively analyze the positive expression rate of the markers to assess cell purity.
[0056] 2.4 Assessment of cell adhesion efficiency
[0057] The adhesion of primary cells in the experimental and control groups after seeding was recorded separately. On day 6 of culture, the cell adhesion rate of the two groups was calculated by cell counting (adhesion rate = number of adherent cells / initial seeded cells × 100%). Three parallel samples were set up for each group, and the average value was taken as the final result to compare and analyze the early survival and adaptability of the two groups of cells.
[0058] 3. Results Analysis
[0059] 3.1 Comparison of primary cell morphology and growth dynamics
[0060] Experimental group (capsule fluid extraction method): such as Figure 1 The diagram shows a simplified procedure of the method of this invention. This method directly obtains cystic fluid containing endothelial cells through puncture, eliminating the need for cumbersome steps such as tissue digestion. Microscopic observation results show ( Figures 5-6 Day 3 of primary cell culture ( Figure 5 That is, typical cobblestone-like or spindle-shaped endothelial cell colonies appear, with uniform cell morphology; cultured to day 6 ( Figure 6 The cells grow in a single layer adhering to the wall like paving stones, with clear cell boundaries, high purity, and almost no interference from impurity cells.
[0061] Control group (traditional tissue separation method): such as Figures 2-4 As shown: On day 3 of primary cell culture, only a small number of cells adhered to the culture vessel, and the adhered cells exhibited diverse morphologies, with a large number of spindle-shaped fibroblast-like cells mixed in; by day 6 of culture ( Figure 3 Although the number of cells increased, the cell morphology remained uneven, and fibroblast-like cells were still present; by day 9 of culture ( Figure 4 Although the cell density reached a high level, there was still significant non-endothelial cell contamination in the cell layer.
[0062] 3.2 Identification results of lymphatic endothelial cell-specific markers
[0063] 3.2.1 Identification by immunofluorescence staining
[0064] like Figure 7As shown, the cells isolated and cultured in the experimental group (capsule extraction method) all highly expressed the classical endothelial cell marker CD31, as well as the lymphatic endothelial cell-specific markers D2-40 (Podoplanin), LYVE-1, and VEGFR-3, confirming from the protein localization level that the obtained cells were lymphatic endothelial cells.
[0065] 3.2.2 Quantitative analysis by flow cytometry
[0066] like Figure 8 (Control group) and Figure 9 Flow cytometry results (for the experimental group) showed that cells obtained by both methods highly expressed CD31 and D2-40; further statistical analysis of the positive rate indicated that... Figures 10-11 The CD31 positive expression rate of cells obtained in the experimental group (capsule fluid extraction method) was 99.14%, which was significantly higher than that of the control group (traditional tissue separation method) at 97.76%, and the difference was statistically significant (p < 0.05), suggesting that the method of the present invention has a stronger enrichment ability for lymphatic endothelial cells.
[0067] 3.3 Comparison of cell adhesion efficiency
[0068] like Figure 12 As shown, the cell adhesion rate on day 6 of primary culture was 97.78% in the experimental group (capsule fluid extraction method), which was significantly higher than 56.47% in the control group (traditional tissue separation method). This indicates that lymphatic endothelial cells derived from capsule fluid have stronger in vitro adhesion ability and early survival viability. The method of this invention can significantly improve the efficiency of early cell growth.
[0069] 4. Conclusion
[0070] This embodiment verifies the feasibility and superiority of the lymphatic endothelial cell cyst fluid extraction method of the present invention. This method is based on... Figure 1 The simplified procedure shown obtains target cells by directly collecting cystic fluid from cystic lymphatic malformations, eliminating the need for complex surgical sampling and tissue digestion steps, making the operation simpler and more efficient. Compared with traditional tissue separation methods, the method of this invention has the following significant advantages:
[0071] (1) Better cell morphology uniformity: The primary cells obtained in the experimental group showed typical lymphatic endothelial cell morphology in the early stage with very little contamination from impurity cells, while the cells in the control group had mixed morphology in the early stage with obvious contamination from non-endothelial cells.
[0072] (2) Higher cell purity: Quantitative analysis by flow cytometry confirmed that the positive rate of endothelial cell marker (CD31) in the experimental group was significantly higher than that in the control group (p<0.05), indicating that the method of the present invention can enrich lymphatic endothelial cells more efficiently and maintain their specific phenotype.
[0073] (3) Stronger early cell growth: The cell adhesion rate of the experimental group was much higher than that of the control group, which confirms that the cells obtained by the method of the present invention have better in vitro adaptability and early proliferation activity.
[0074] In summary, the cyst fluid extraction method of this invention is an efficient, simple, and reliable method for isolating and culturing lymphatic endothelial cells in lymphatic malformations. It can effectively solve many drawbacks of traditional methods and provide a stable and high-quality cell model for basic experiments such as the study of the pathogenesis of lymphatic malformations and drug screening. It has important clinical application value and scientific research prospects.
[0075] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
Claims
1. A method for extracting cystic fluid from lymphatic endothelial cells in lymphatic vessels with malformations, characterized in that, Includes the following steps: S1. Sample collection: Collect 5-10 mL of cystic fluid from patients with large cystic lymphatic malformations and place it in a sterile EP tube or centrifuge tube. S2. Initial centrifugation: Use low-speed centrifugation. After centrifugation, discard the supernatant and retain the cell pellet at the bottom. S3, Red blood cell lysis: Add an appropriate amount of red blood cell lysis buffer to the cell pellet described in step S2, mix well, and place at 4°C for an appropriate time. After placement, centrifuge, discard the supernatant, wash with PBS, and centrifuge again. S4. Cell Culture: Resuspend the cell pellet treated in step S3 in a culture medium containing endothelial growth factor supplement, seed it into 6-well plates or culture flasks, and incubate in an incubator; after 24 hours of culture, change the culture medium and remove non-adherent cells. S5 cell identification: Immunofluorescence staining can be performed to identify cells 48-72 hours after cell adhesion.
2. The method for extracting endothelial cell vesicle fluid from lymphatic vessels with malformations according to claim 1, characterized in that, In step S1, the cyst fluid is collected using a sterile syringe.
3. The method for extracting endothelial cell vesicle fluid from lymphatic vessels with malformations according to claim 1, characterized in that, The low-speed centrifugation speed in step S2 is 1000 rpm, and the centrifugation time is 10 minutes.
4. The method for extracting endothelial cell vesicle fluid from lymphatic vessels with malformations according to claim 1, characterized in that, The appropriate time in step S3 is 3 to 5 minutes; the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes.
5. The method for extracting endothelial cell vesicle fluid from lymphatic vessels with malformations according to claim 1, characterized in that, The conditions for incubation in step S4 are 37°C and 5% CO2.