CN-122012391-A - Lymphatic endothelial cell cyst fluid extraction method for lymphatic malformation

Abstract

The invention discloses a lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method, and belongs to the technical field of biological cell extraction. The method comprises the steps of S1, collecting samples, namely 5-10 mL of cyst fluid of a patient with large cystic lymphatic malformation is collected and placed in a sterile container, S2, primary centrifugation, namely low-speed centrifugation at 1000rpm for 10 minutes, cell precipitation is reserved, S3, erythrocyte lysis, namely adding erythrocyte lysate, placing at 4 ℃ for 3-5 minutes, centrifuging, washing and purifying by PBS, S4, cell culture, namely resuspending cells in a culture medium containing endothelial growth factor supplementing liquid, culturing at 37 ℃ and 5% CO 2 , changing liquid for 24 hours to remove non-adherent cells, and S5, cell identification, namely immunofluorescence staining identification after cell adherence for 48-72 hours. The method is simple and efficient in operation, does not need complex digestion, can enrich the lymphatic endothelial cells with high purity and high activity, provides a stable cell model for relevant pathogenesis research and drug screening, and has important scientific research and clinical value.

Inventors

• JI YI
• XIANG PANPAN
• Xing Rongjing
• QIU TONG
• ZHOU ZILONG
• ZHANG ZIXIN
• LAN Yuru
• Zhou Jiangyuan
• GONG XUE
• ZHANG YUJIA
• CHEN SIYUAN

Assignees

• 四川大学华西医院

Dates

Publication Date

20260512

Application Date

20260112

Claims (5)

1. 1. A lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method, which is characterized by comprising the following steps: S1, collecting samples, namely collecting 5-10 mL of cyst fluid of a patient with large cystic lymphatic vessel malformation, and placing the cyst fluid in a sterile EP tube or a centrifuge tube; S2, primary centrifugation, namely adopting low-speed centrifugation, discarding supernatant after centrifugation, and reserving bottom cell sediment; S3, erythrocyte lysis, namely adding a proper amount of erythrocyte lysate into the cell sediment in the step S2, uniformly mixing, standing at 4 ℃ for a proper time, centrifuging after standing, washing with PBS (phosphate buffer solution) after removing the supernatant, and centrifuging again; S4, cell culture, namely re-suspending the cell sediment treated in the step S3 in a culture medium containing endothelial growth factor supplementing liquid, inoculating the cell sediment in a 6-hole plate or a culture bottle, placing the cell sediment in a culture box for culture, replacing the culture medium after culturing for 24 hours, and removing non-adherent cells; s5, identifying the cells, namely identifying the cells by immunofluorescence staining 48-72 hours after cell adhesion.
2. 2. The lymphatic malformed lymphatic endothelial cell cyst fluid extraction method of claim 1, wherein said cyst fluid is collected in step S1 using a sterile syringe.
3. 3. The lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method according to claim 1, wherein said low speed centrifugation speed in step S2 is 1000rpm and centrifugation time is 10 minutes.
4. 4. The lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method according to claim 1, wherein in step S3, the suitable time is 3-5 minutes, the re-centrifugation speed is 1000rpm, and the centrifugation time is 5 minutes.
5. 5. The lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method according to claim 1, wherein said culturing conditions in incubator in step S4 are 37 ℃ and 5% co 2 .

Description

Lymphatic endothelial cell cyst fluid extraction method for lymphatic malformation Technical Field The invention relates to the technical field of biological cell extraction, in particular to a lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method. Background Lymphatic malformations (LYMPHATIC MALFORMATION, LM) are vascular diseases formed by dysplastic lymphatic vessels, commonly found in children's neck and face, armpit, mediastinum, etc. The capsule cavity often contains a large amount of transparent capsule liquid, and the capsule liquid is rich in exfoliated lymphatic endothelial cells, inflammatory cells and various bioactive substances. However, at present, cytology and molecular research on LM focus are mostly dependent on operation excision tissue to carry out primary cell separation culture, the process has larger trauma to the infant, the sample is difficult to obtain, and more fiber tissue and erythrocyte pollution exist in the operation sample, so that the purification difficulty is high, the cell activity is low, and the culture success rate is unstable. Disclosure of Invention The invention aims to provide a lymphatic endothelial cell cyst fluid extraction method for lymphatic malformation of lymphatic vessels, which solves the problems of complicated steps, low cell purity, poor early growth activity and the like in the traditional tissue separation method and provides a high-quality cell model for the relevant basic research and clinical transformation of lymphatic vessel malformation. The invention provides a lymphatic vessel malformed lymphatic endothelial cell cyst fluid extraction method, which comprises the following steps: S1, collecting samples, namely collecting 5-10 mL of cyst fluid of a patient with large cystic lymphatic vessel malformation, and placing the cyst fluid in a sterile EP tube or a centrifuge tube; S2, primary centrifugation, namely adopting low-speed centrifugation, discarding supernatant after centrifugation, and reserving bottom cell sediment; S3, erythrocyte lysis, namely adding a proper amount of erythrocyte lysate into the cell sediment in the step S2, uniformly mixing, standing at 4 ℃ for a proper time, centrifuging after standing, washing with PBS (phosphate buffer solution) after removing the supernatant, and centrifuging again; S4, cell culture, namely re-suspending the cell sediment treated in the step S3 in a culture medium containing endothelial growth factor supplementing liquid, inoculating the cell sediment in a 6-hole plate or a culture bottle, placing the cell sediment in a culture box for culture, replacing the culture medium after culturing for 24 hours, and removing non-adherent cells; s5, identifying the cells, namely identifying the cells by immunofluorescence staining 48-72 hours after cell adhesion. Further, in step S1, a sterile syringe is used to collect the cyst fluid. Further, the low-speed centrifugation speed in step S2 is 1000rpm, and the centrifugation time is 10 minutes. Further, in the step S3, the proper time is 3-5 minutes, the re-centrifugation rotating speed is 1000rpm, and the centrifugation time is 5 minutes. Further, the conditions for culturing in the incubator in step S4 are 37 ℃ and 5% co 2. Compared with the prior art, the invention has the beneficial effects that: (1) The trauma is lower, only the cyst fluid sample is required to be extracted, the tissue is not required to be resected by operation, and the damage risk of the patient is greatly reduced. (2) Simple operation and short time, no need of collagenase and pancreatin digestion, and the primary separation of cells can be completed within 30 minutes by a single operation. (3) The cell purity is higher, the endothelial cells from the cyst fluid are relatively rich, the pollution is less after the erythrocyte is lysed, and the endothelial markers (such as CD31, LYVE-1, D2-40 and VEGFR 3) are expressed stably. (4) The culture success rate is high, the cell source activity is good, the adherence is fast, the morphology is uniform, and the method is suitable for the subsequent immunofluorescence, molecular experiments and drug sensitivity detection. (5) The repeatability and the popularization are strong, the requirements on the sample size, the cyst fluid source and the experimental conditions are low, and the method is suitable for multi-center sample standardized operation. Drawings FIG. 1 is a schematic flow chart of a lymphatic endothelial cell cyst fluid extraction method for lymphatic malformation according to an embodiment of the present application; FIG. 2 is a schematic diagram showing microscopic observations on day 3 of primary culture by LMLEC conventional tissue isolation; FIG. 3 is a schematic diagram of microscopic observations on day 6 of primary culture in LMLEC conventional tissue isolation; FIG. 4 is a schematic diagram of microscopic observations on day 9 of primary culture in LMLEC conventional tissue isolation; FIG. 5