Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

The overall goal of this procedure is to isolate high purity lymphatic endothelial cells that line the lymphatic vessels of human lymphatic malformations and foreskins using fluorescence activated cell sorting. This is accomplished first by enzymatically digesting a patient tissue sample. In the second step, the single cell suspension is cultured to enrich the number of lymphatic endothelial cells.

In the sample. The cells are then labeled with antibodies to the cell surface markers of interest and sorted using multi-parameter fluorescence activated cell sorting. Ultimately, the foreskin and lymphatic malformation, lymphatic endothelial cells can be expanded in cell culture for further downstream analysis.

The main advantage of this technique over existing methods is that fluorescence activated cell sorting yields cells are greater than 99%purity, avoiding the problems associated with the loss of lymphatic endothelial cells due to overgrowth by contaminating cells. Begin by weighing a 50 milliliter tube containing 10 milliliters of medium, supplemented with an antibiotic antimycotic solution. Then transfer the lymphatic malformation or four skin sample into the tube and weigh the tube again to determine the weight of the tissue.

Next, in a class two biosafety cabinet, use sterile forceps to transfer the tissue and medium into a 100 millimeter tissue culture dish using sterile scissors. Finally, mince the tissue into one cubic millimeter pieces. Then transfer the pieces into a 50 milliliter tube with 10 milliliters of fresh antibiotic antimycotic.Medium.

Swirl the tube a few times to resuspend the tissue and then spin down the sample. After removing the supernatant, add one milliliter of prewarm digestion solution per 100 milligrams of minced tissue and incubate the tissue in a 37 degree Celsius incubator with constant shaking at 200 RPM For successful enrichment of the lymphatic endothelial cells. It is critical to recognize when the cells have been sufficiently exposed to the collagenase and dys space reaction so that they survive the isolation.

This is achieved through careful monitoring of the tissue digestion process and stopping the reaction when most of the tissues has been digested into fine fragments. At the end of the incubation, confirm that nearly all of the tissue has digested into small fragments and filter the tissue slurry through a 70 micron strainer into a sterile 50 milliliter tube. Now use a three milliliter syringe piston with rubber plunger to grind down the remaining tissue until only small traces of extracellular matrix are observed.

Then wash the cell strainer with a volume of endothelial cell medium equivalent to the volume of the dissociating enzyme medium and spin down the cells reus. Suspend the pellet in up to 20 milliliters of endothelial cell medium, depending on the specimen size. Then after counting seed, two times 10 to the six cells in a 150 square centimeter flask pre-coded with fibronectin in a final volume of 20 milliliters of endothelial cell medium for overnight culture.

The next morning, wash away the unbound cells with three rinses of PBS supplemented with antibiotic antimycotic solution. Then to expand the number of lymphatic endothelial cells, add fresh endothelial cell medium to the cells, and incubate the culture for five to seven days until the culture is about 80%confluent. To stain the cells for sorting by multi-parameter fluorescence activated cell sorting.

First, remove the cell media, wash the cells three times in PBS. Then add seven milliliters of detachment solution after five to seven minute incubation at 37 degrees Celsius. Harvest the dissociated cells, inactivate the enzymes with three volumes of endothelial cell medium, and transfer the cell suspension into a sterile tube.

Centrifuge the cells three times washing the pellet in five milliliters of PBS for the second and third spins after the last wash. Resuspend the pellet in five milliliters of fresh PBS and count the number of viable cells by trian blue exclusion. Following cell counting.

Aseptically transfer the cells to flow cytometry staining tubes, and spin down the cells to block nonspecific binding. Resuspend the cells in 5%F-B-S-P-B-S, and incubate on ice for 20 minutes. After blocking centrifuge, the cells remove the supernatant and resus.

Suspend the pellet in CD 34, CD 31 potanin and VEGF receptor three antibody cocktail. After 20 minutes on ice, wash the cells three times in two milliliters of F-B-S-P-B-S to remove any unbound antibody and resus. Suspend the pellet in 300 microliters of propidium iodide in F-B-S-P-B-S solution on ice.

Finally, sort the purified human lymphatic endothelial cells on a multi-parameter fluorescence activated cell sorting instrument. Then spin down the sorted cells and resus suspend the pellets in the appropriate medium for cell culture seeding. Here, normal human lymphatic and lymphatic malformation vessels labeled with antibody against pot deplane in are shown note the marked dilation and considerable lumen size variation within the human lymphatic malformation vessels.

Indeed, most lymphatic malformations contain both small or microcystic and large or macrocystic abnormal cystic structures following the initial tissue digestion and 24 hours of culture in the unfractionated samples. Distinct endothelial cell colonies can be observed in addition to the fibroblast like cells and smooth muscle cells after sorting and a further 24 hours in cell culture. However, the CD 34, low CD 31 positive VEGF receptor three positive podo plane and positive cells attached to the culture container and exhibit the typical cobblestone morphology observed in these images.

After watching this video, you should have a good understanding of how to isolate with high purity the lymphatic endothelial cell that lines human lymphatic malformation and full skin lymphatic vessels using fluorescence activated cell sorting. Don't forget that working with primary human tissue can be hazardous as it may contain infectious agents harmful to human health. Standard precautions such as wearing gloves, protective eyewear, and laboratory gown should always be taken while performing this procedure.