Isolation of Human Lymphatic Endothelial Cells

Materials and Reagents

All reagents are stored and prepared as per manufacturer’s instructions. Prepare enzyme solutions on the day of cell isolation. Warm the endothelial cell media, cell culture buffers, and enzyme solutions at 37 ℃ for 30 min prior to use. All solutions stored at 4 ℃ as per manufacturer’s instructions should be warmed to room temperature only for the time needed to use them. All materials should be disposed of after use as specified by one’s own institutional safety guidelines.

Culture Medium and Solutions

1.Endothelial cell media: EGM-2 MV Bullet Kit (Lonza, catalogue numbercc-3202) supplemented with 50 ng/mL VEGF-C (R&D, 2179-VC-025). The media is prepared by warming EGM-2 media and gently thawing the kit components. Once thawed, the individual components are added to 500 mL EGM-2 media bottle in the laminar flow cell culture hood. Add VEGF-C last. Aliquot endothelial cell media into 50 mL sterile tubes, and store at 4 ℃ until use.

2.Calcium and magnesium-free phosphate-buffered saline (PBS): Prepare PBS by dissolving 8.752 g NaCl, 1.416 g Na₂HPO₄2H₂O, and 0.395 g KH₂PO₄in 1000 mL of water. Adjust pH to 7.4. Filter sterilize PBS and store at 4 °C. Prior to use, add 100 U/mL penicillin and streptomycin.

3.Human fibronectin (Sigma-Aldrich, catalogue number F2006): Dissolve 1 mg of fibronectin in 1 mLof sterile H₂ Store reconstituted solution in 100 μL aliquots at −20 °C. On the day of use, add 10 mL of PBS to 100 μL of fibronectin aliquot (to give a final working concentration of 10 μg/mL). For 25 cm² flasks, use 1 mL of fibronectin solution to coat the flask; for 75 cm², use 4 mL to coat the flask; and for 150 cm², use 7 mL to coat the flask. Note that 10 μg/mL fibronectin solution can be reused as the sterile reconstituted solution is stable at 4 °C for 1 month.

4.StemPro ® Accutase ® Cell Dissociation Reagent (Life Technologies, Gibco ®, catalogue number A11105-01): This reagent is used to detach cells during passaging as it preserves CD34 and CD31 expression and cell viability better than trypsin-based detachment agents.

5.4 % trypan blue solution (Life Technologies, Gibco®, catalogue number 15250-061). Tocount cells, 10 μL of cell suspension is mixed with 90 μL of trypan blue. 10 μL of this suspension is used in hemocytometer counting.

6.Dimethyl sulfoxide (DMSO, Sigma Aldrich, catalogue number D4540).

Enzymatic Digestion

The following enzymes are required: Dispase II (Roche Applied Biosciences, catalogue number 4942078001), Collagenase Type II (Worthington Lab, catalogue number 4176) and DNAse I(Roche Applied Biosciences, catalogue number 11284932001). Based on tissue weight, prepare an appropriate volume of enzyme media containing 0.04 % dispase II, 0.25 % collagenase II, and 0.01 % DNase I in sterile  Ten mL of enzymatic media is required per 1 g of tissue. First, weigh required weight of dispase and collagenase into a sterile 50 mL tube, then add the required volume of PBS, and incubate for 30 min with shaking at 37 °C to dissolve. Once dissolved, filter sterilize (0.22 μm filter) dispase/collagenase solution, and in the laminar fl ow hood, aseptically add the required amount of DNase I. The enzymatic media is always prepared on the day of use and kept at 37 °C until use.

Magnetic Bead Cell Isolation

In addition to columns (see equipment), the following Miltenyi Biotec reagents are required:CD31 Multisort kit (catalogue number 130-091-935), CD34 Multisort kit (catalogue number 130- 056-701), anti-fibroblast beads (catalogue number: 130-050-601), MACS BSA stock solution (catalogue number 130-091-376), and MACS rinse buffer (catalogue number 130-091-222). Aseptically combine MACS BSA stock solution and MACS rinse buffer prior to use. Avoid causing bubbles, and degas the solution to remove existing air bubbles and store at 4 °C.

Equipment

All procedures are done aseptically in a Class II laminar flow safety hood equipped with UV light for decontamination. Dissection equipment should be thoroughly washed, cleaned in 70 % ethanol, and autoclaved at 121 °C for 20–30 min (depending on the system used).

1. 1. 25 cm²and 75 cm² tissue culture flasks and 90 mm petri dishes(Ucallm).
   2. Sterile 5 mL and 10 mL tissue culture pipettes(Ucallm).
   3. 15 and 50 mL sterile centrifuge tubes(Ucallm).
   4. 100 μm and 70 μm cell strainers (Ucallm).
   5. 22 μm filtration membranes for solutions(Ucallm).
   6. Vacuum pump to aid filtration process.
   7. 3 mL sterile syringe plunger with black rubber tip.
   8. Scalpel handle, scalpel blades, forceps, and small scissors with straight ends.
   9. Hemocytometer.
   10. Water bath or incubator with a shaker and temperature control.
   11. MACS MultiStand (Miltenyi Biotec).
   12. MidiMACS Separator (Miltenyi Biotec).
   13. MACS LS Columns (Miltenyi Biotec).
   14. Centrifuge suitable to hold 15 mL and 50 mL tubes.
   15. Cell culture incubator with temperature and gas composition control.
   16. Phase contrast microscope and bright field microscope.
   17. Plastic container for cell staining(Ucallm).

Procedure

1.Tissue Collection

Prior to collecting the tissue specimen, pre-weigh sterile container with medium into which the specimen will be collected. Foreskin tissues are collected at the time of surgery into a sterile container containing known amount of endothelial cell media and transported to the laboratory on ice. The amount of media used depends on the tissue size, ensuring that the tissue specimen is completely covered. The tissue is transported to the laboratory on ice and processed immediately. In the laboratory, weigh the container with the specimen, and then subtract the weight of the empty container and the amount of media in the container to obtain tissue weight. Add 1 mL of enzyme media per 100 mg of tissue.

2.Tissue Processing and Cell Isolation

1)Decontaminate thelaminar hood with UV light and clean the working space with 70 % ethanol.

2)Transfer tissue into a 90 mm petri dish, and using sterile forceps and scalpel (or scissors),mince the tissue into 1–2 mm  This is a critical step as better enzyme digestion is achieved with finely minced tissue ( see Notes 1 and 2).

3)Transfer minced tissue aseptically into a sterile 50 mL tube, and add 1 mL of enzyme media (0.04 % dispase II, 0.25 % collagenase II, and 0.01 % DNase I) per 100 mg of tissue. Place the tube at 37 °C with shaking. Neonatal foreskin samples require approximately 30 min. To judge if the incubation is sufficient, simply examine the solution for the amount of undigested tissue present.

4)Following incubation, pass the cells through a 100 μm cell strainerplaced in a sterile 50 mL tube, and then use a sterile 3 mL syringe black with rubber plunger to grind down the remaining tissue until only traces of extracellular matrix remain.

5)Wash the cell strainer with 10 mL of endothelial cell media (1:1 ratio to enzyme media used) to recover cells attached to the sieve and to inactivate the enzymes in cell suspension.

6)Spin the cells at 300 × g for 5 min, remove the supernatant, then wash the cells three times in sterile PBS/PenStrep, each time discarding the supernatant and gently disrupting the cell pellet prior to a new wash.

7)Add endothelial cell media to resuspended cells (5 mL for 25 cm²flask, 10 mL per 75 cm² flask, and 20 mL for 150 cm² flask), and pass the suspension through a 70 μm strainer to remove cell debris. Seed up to 5 × 10⁵ cells in a 25 cm² flask, 2 × 10⁶ cells per 75 cm² flask, and 5 × 10⁶ cells in 150 cm² flasks.

8)Plate the cells in flasks and incubate at 37 °C, 5 % CO₂, and 21 % O₂in a humidified incubator. Check cells after 24 h to determine cell attachment.

9)After 24 h, wash away unbound cells (3 × 5 min PBS/PenStrep) and add new endothelialcell media. Return to the incubator. Change the media every second day ( see Note 3). When cells are approximately 80 % confluent, cell-specific isolations can commence.

3.Magnetic Bead Cell Selection

We follow the Miltenyi Biotec manufacturer’s instruction with this procedure. However, in addition, we repeat purification on a fibroblast and CD34 column twice to ensure maximum removal of fibroblasts and CD34^Pos cells from the cell suspension.

4.Fibroblast Depletion

1)Remove the media from the flask and wash the cells three times with sterilePBS/PenStrep. To prepare the cell suspensions, add pre-warmed Accutase ® to each flask. For 25 cm² flask use 1 mL, for 75 cm² use 5 mL, and for 150 cm² use 7 mL to coat the flask. Generously cover the cells with Accutase ® to ensure complete cell detachment from the fibronectin -coated flask.The cells are incubated for 5 min (maximum 7 min) at 37 °C in Accutase ®. Examine under the polarized microscope, and if the cells are not completely detached, carefully tap the flask three to four times to detach them.

2)Add endothelial cell media to the flask (3:1 ratio of media to Accutase ®) to inactivateAccutase ®. The cell suspension is drawn up and down a sterile cell culture pipette three to four times before transfer to a new sterile 15 mL centrifuge tube.

3)Centrifuge the cells at 300 × g for 5 min, remove the supernatant, and resuspend the cells in 2 mL of endothelial cell media. Count the cells.

4)To deplete fibroblasts from the cell suspension, centrifuge the suspension at 300 × g for 5 Aspirate the supernatant completely. Resuspend the cell pellet in 80 μL of MACS buffer per 10⁷ total cells, and avoid introducing air bubbles as this slows down the cell isolation.

5)Add 20 μL of anti-fibroblast beads per 10⁷ total cells. Mix well with a pipette and incubate for 30 min at room temperature. During this time, prepare the LS column by inserting it into the magnetic field separator. Place a new 15 mL sterile tube at the tip of the column to collect the rinse buffer. Rinse the column with 3 mL of MACS buffer to prepare the column for use. Place a new 15 mL sterile tube at the tip of the column to collect the flow through. The columns will not dry out while you are waiting for the incubation to be completed.

6)At the end of incubation, add 2 mL of MACS bufferper 10⁷ cells to wash away unbound beads and centrifuge at 300 × g for 10 min. Aspirate supernatant completely then resuspend cells in 500 μL of MACS buffer.

7)Apply the cell suspension to the column. Do not introduce air bubbles as this will either block the column and the sample will be lost, or it will slow the elution and prolong the isolation The flow through is collected as it contains fibroblast depleted cells. Once the flow has ceased, wash the column three times with MACS buffer. Continue to collect the flow through.

8)On the final wash, place the flow through into the centrifuge, spin at 300 × g for 5 min, and then repeat the process for cell suspension and column purification. This step will minimize the fibroblast contamination. Again collect the flow through, centrifuge and remove thesupernatant, and resuspend in cell media to count the cells. Depending on the tissue sample, typical yield is 2 × 10⁵–5 × 10⁵ cells at this stage. To prepare the cells for CD34 depletion, centrifuge and remove the supernatant.

5.CD34 Depletion

1)Resuspend the cell pellet in 80 μL of buffer then add 20 μL of Anti- CD34 MicroBeads.Mix well with a 100 μL pipette, then incubate at 2–8 °C for 30 min. During this time prepare the column for CD34 separation as described above.

2)At the completion of incubation, add 2 mL of MACS buffer to the cells to facilitate removal ofthe unbound beads then centrifuge at 300 × g for 5 min.

3)Remove the supernatant and add 500 μL of MACS buffer to the cell pellet. Gentlyresuspend cells with 1 mL pipette and load the cells onto the column to deplete CD34^Pos Collect the flow through.

4)Once the cell suspension has gone through the column, wash the column three times using 3 mL of MACS buffer, each time continuing to collect the flow through. The flow through contains CD34^Neg

5)Once the CD34^Negcell fraction is collected, centrifuge the cells at 300 × g, remove the supernatant, resuspend the cells in 1 mL of buffer, and then repeat the column purification to remove any remaining CD34^Pos cells that might have “squeezed” through. This step further reduces CD34^Pos cells, which also include CD34^Pos nonvascular cells.

6)After the second column purification, centrifuge the CD34^Negnegative fraction cells at 300 × g for 5 min, remove the supernatant, and proceed with CD31 purification. At this stage, the cell yield is about 10,000–50,000 cells (depending on the initial specimen size).

6.CD31 Positive Selection on Fibroblast and CD34-Depleted Cells

1)Following CD34 depletion, resuspend the cell pellet in 80 μL of MACS bufferusing 100 μL pipette being careful not to introduce air bubbles.

2)Add 20 μL of Anti- CD31 MicroBeads. Mix well with pipette then incubate at2–8 °C for 15 min. During this time prepare the MACS column as described above.

3)After the incubation is completed, add 1 mL ofMACS buffer to the cells to wash unbound beads then centrifuge at 300 × g for 5 min.

4)Remove the supernatant and resuspend the cells in 500 μL of MACS buffer prior to loading them onto the column to collect CD31^Pos This time, the flow through is not needed since when examined microscopically there are too few cells in the flow through to warrant an additional column isolation. Once you have loaded the cells on to the column, wash the column three times using 3 mL of MACS buffer each time.

5)Once the CD31^Negfraction has been collected, remove the column from the separator and place it on a new 15 mL steriletube to collect cells. Pipette 5 mL of buffer onto the column, and using the column plunger provided, immediately flush out the magnetically labeled cells (CD31^Pos fraction) by firmly and Quickly pushing the plunger into the column.

6)Centrifuge the cells at 300 × g, remove the supernatant, then gently resuspend the cells in endothelial cell media,Count cells, then are used for downstream application.

7.Notes

1)The LEC yield will depend on foreskin age. Neonatal skin aged <3 months givesa greater cell yield than foreskin aged >6 months.

2)Always prepare fresh enzyme media as this works better when the tissue is more fibrotic.

3)Change the media every second day as longer times between media changesfavor survival of fibroblasts and/ or vascular smooth muscle cells, even if they are present in small quantities (2–5 %).

References

1. Lokmic, Z. (2016). Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells. In: Martin, S., Hewett, P. (eds) Angiogenesis Protocols. Methods in Molecular Biology, vol 1430. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3628-1_5

2.Hirakawa S, Hong YK, Harvey N, Schacht V, Matsuda K, Libermann T et al (2003) Identifi cation of vascular lineage-specifi c genes by transcriptional profi ling of isolated blood vascular and lymphatic endothelial cells. Am J Pathol 162(2):575–586.

3.Lokmic Z, Ng ES, Burton M, Stanley EG, Penington AJ, Elefanty AG. Isolation of human lymphatic  endothelial cells by multi-parameter fluorescence-activated cell sorting. J Vis Exp. 2015 May 1;(99):e52691. doi: 10.3791/52691. PMID: 25992474; PMCID: PMC4652192.

Manufacturer’s Link

R&D Systems: https://www.rndsystems.com

Sigma-Aldrich: https://www.sigmaaldrich.com

Thermo Fisher Scientific: https://www.thermofisher.com›home›brands ›gibco

Roche: https://lifescience.roche.com ›global

Worthington Lab: https://worthington.lab.uiowa.edu

Miltenyi Biotec: https://www.miltenyibiotec.com

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