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Endothelial Cell Isolation Protocol

Endothelial cells (ECs) demonstrate unique phenotypic and functional characteristics specific to each tissue, essential for research in vascular biology and other fields. Therefore, it is important to employ tissue-specific techniques for isolating highly purified ECs. Here is a basic protocol for isolating endothelial cells from tissue samples. This protocol can be applied to various tissues, but specific steps might need to be adjusted based on the tissue type.

Tab.1 The essential materials for isolating and culturing ECs

Materials Needed Description
Enzymes Collagenase, trypsin, or dispase for tissue digestion and cell dissociation.
PBS For washing tissues and cells.
Trypsin-EDTA solution For detaching cells from culture vessels.
Culture medium Specific endothelial cell growth medium, often supplemented with growth factors.

Preparation

Sterilize all tools and work surfaces.

Pre-warm the collagenase solution, trypsin-EDTA, and endothelial cell growth medium to 37°C.

  1. Tissue Harvesting
    Dissect the tissue of interest under aseptic conditions.
    Rinse the tissue in cold PBS to remove blood and debris.
  2. Tissue Mincing
    Mince the tissue into small pieces (1-2 mm3) using sterilized scissors or a scalpel.
  3. Enzymatic Digestion
    Transfer the minced tissue into a sterile tube containing pre-warmed collagenase solution.
    Incubate the tissue in a water bath or incubator at 37°C for 30-60 minutes with gentle agitation. (The duration may vary depending on the tissue type and collagenase concentration.)
    Optionally, add DNase I to the collagenase solution if the tissue has a high DNA content to prevent cell clumping.
  4. Mechanical Dissociation
    After digestion, gently pipette the mixture up and down to dissociate the cells further.
    Filter the cell suspension through a 70 µm mesh cell strainer into a fresh sterile tube to eliminate any remaining tissue debris.
  5. Centrifugation
    Centrifuge the cell suspension at 300 x g for 5-10 minutes to collect the cells into a pellet.
    Discard the supernatant and carefully resuspend the cell pellet in PBS or growth medium.
  6. Trypsinization
    If further dissociation is needed, treat the cell suspension with trypsin-EDTA for 5-10 minutes at 37°C.
    Add an equivalent amount of growth medium supplemented with fetal bovine serum (FBS) to deactivate trypsin.
  7. Cell Plating
    Resuspend the cells in the endothelial cell growth medium.
    If needed, plate the cells in a cell culture flask or dish pre-coated with fibronectin or gelatin.
    Culture the cells at 37°C in a humidified atmosphere containing 5% CO2.
  8. Cell Culture
    Replace the medium after 4-6 hours to eliminate non-adherent cells.
    Replace the medium every 2-3 days until the cells reach confluency.
  9. Purification (optional)
    To purify endothelial cells, perform magnetic or flow cytometric cell sorting using endothelial cell-specific markers such as CD31 or VE-cadherin.

Notes

  1. Depending on the tissue type, the specific concentration of collagenase and digestion time may need optimization.
  2. Maintaining sterile conditions and working efficiently are crucial to minimize the possibility of contamination.
  3. Regularly monitor the cells under a microscope to assess their morphology and growth.

SERVICES

Creative Biolabs provides a comprehensive and detailed protocol for isolating endothelial cells from tissue samples. This protocol is designed to ensure the efficient and high-quality extraction of endothelial cells, critical for various research applications, including vascular biology, cancer research, and regenerative medicine.


All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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