hsatmvec-adipocyte co-culturing to investigate cellular cross talks in patients undergoing cied implantation 

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Endothelial cells (ECs) are squamous cells that line the inner surface of the blood vessel wall as a monolayer. They have many essential roles, including control of vascular tone, regulation of thrombosis, modulating the inflammatory response, and contributing to angiogenesis1. Given the importance of endothelial cells in cardiometabolic physiology, they are frequently used experimentally to further the understanding of pathophysiology and to examine new pharmacological treatments for cardiometabolic disease.
However, there is enormous heterogeneity in endothelial cell morphology, function, gene expression, and antigen composition depending on the origin of their vascular bed2. While endothelial cells from large arteries are best suited to atherosclerosis studies, endothelial cells from small vessels, known as microvascular endothelial cells (MVECs), are more suitable for angiogenesis studies2. Understanding the molecular basis for endothelial heterogeneity may provide valuable insights into vascular bed-specific therapies. Microvascular endothelial function also significantly differs in numerous diseases, including diabetes, cardiovascular disease, and systemic infection3,4. Therefore, the ability to isolate endothelial cells from defined patient groups allows direct comparison of their endothelial cell function and cellular cross-talk5. 
In this paper, we describe a novel method of isolating human MVECs from subcutaneous adipose tissue (hSATMVEC) taken at the time of cardiac implantable electronic device (CIED) insertion. hSATMVEC isolated following enzymatic digestion of subcutaneous adipose tissue (SAT) were grown and passaged on gelatin-coated plates. We then describe a range of phenotyping assays that have been successfully applied to hSATMVECs in order to validate their phenotype and demonstrate use in routine endothelial cell assays. Finally, we describe an application of hSATMVECs in an experimental model of hSATMVECs-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

We want to understand the role of the subcutaneous adipose tissue microvasculature in people with and without cardiometabolic disease. To do this, we've been isolating subcutaneous adipose tissue microvascular endothelial cells from patients undergoing cardiac implantable electronic devices. This is to study the endothelial cell phenotype and cell-cell crosstalk with adipocytes.Historically, we have used immortalized human cell lines or animal models to study cardiometabolic disease in vitro. However, these are not representative of diseases in human subjects. Our technique overcomes this by allowing us to understand the cellular basis of cardiometabolic disease using tissue from real-world patients.Our group is currently investigating the mechanism of dysregulated subcutaneous adipose tissue endothelial cell adipocyte crosstalk in people with diabetes, in particular, the impact of diabetic endothelium on adipocyte function and how this can be targeted to improve cardiometabolic health.

hSATMVEC-Adipocyte Co-Culturing to Investigate Cellular Cross Talks in Patients Undergoing CIED Implantation 

To begin, thaw the passage, one human white subcutaneous preadipocytes, and add the cells into a T75 flask containing 12 to 15 milliliters of warm preadipocyte growth media-2. After the cells attain 90%confluency, wash them with PBS and incubate them with one milliliter of 0.25%Trypsin-EDTA solution for two minutes. Using a light microscope, confirm the cell detachment and add 23 milliliters of preadipocyte growth media-2 to the cells.Dispense one milliliter of cell suspension into each well of a 24 well co-culture companion plate and incubate the plate to attain cell confluency. Then replace the media with one milliliter of preadipocyte differentiation medium and incubate at 37 degrees celsius with 5%carbon dioxide for 10 days. On day six of differentiation, seed five times 10 to the power of four human SAT MVECs in 500 microliters of complete MVEC growth media on a transwell insert.Place the insert in a well containing 500 microliters of complete MVEC growth media and incubate at 37 degrees Celsius with 5%carbon dioxide. On day 10, when the adipocytes are fully differentiated, remove half the media from each well and transfer the transwell inserts containing confluence SAT MVEVs to each well. Incubate the plate for 24 hours to obtain a co-culture.As the human subcutaneous white preadipocytes become more differentiated, the development of lipid vacuoles can be noticed.

hsatmvec-adipocyte-co-culturing-to-investigate-cellular-cross-talks

Research

JoVE Journal

Medicine

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics

To begin, obtain 250 milligrams of SAT above the pectorals major muscle during CIED implantation in magnetic-activated cell-sorting tissue storage solution. Prepare one milligram per milliliter of Collagenase/Dispase in 10 milliliters of cold Hanks'Balanced Salt Solution. Under a laminar flow cabinet, mince tissues into one-cubic millimeter pieces in 500 microliters of Collagenase/Dispase solution.Add 4.5 milliliters of the Collagenase/Dispase solution to the mix and transfer the triturated tissue to a clean 50-milliliter centrifuge tube. Wash the Petri dish lid with five milliliters of Collagenase/Dispase solution and add it to the tube. Incubate the tube for 30 minutes at 37 degrees Celsius in a tube rotator set to 20 revolutions per minute.To halt the digestion, pour 10 milliliters of complete endothelial cell growth media into the tube. After triturating the digested mix with a 14-gauge Venflon cannula, strain it through a 70-micrometer cell sieve and rinse it with 10 milliliters of 0.5%PBS-BSA buffer. Centrifuge the filtrate at 300G for 10 minutes at room temperature and discard the supernatant.To remove the dead cells, add 200 microliters of dead cell removal beads to the cell pellet taken in a microcentrifuge tube, and incubate. Then attach a liquid-separation column with a 30-micrometer filter to the separator magnet and place a 15-milliliter centrifuge tube underneath. After washing the column with dead cell removal binding buffer, add the sample bead suspension.Let it pass through under gravity and collect the eluate. Next, spin the collected live cell eluate and resuspend the resulting pellet in 400 microliters of 0.5%PBS-BSA. Incubate the suspended cells with 20 microliters of anti-CD31-coated magnetic beads at four degrees Celsius for 20 minutes.After centrifuging, resuspend the cell pellet in 500 microliters of 0.5%PBS-BSA. Attach a column with a 30-micrometer filter to the separator magnet and place a 15-milliliter centrifuge tube underneath. Add the cell microbead suspension to the column and let it pass through under gravity.After washing the column, place it in a fresh 15-milliliter centrifuge tube and add one milliliter of 0.5%PBS-BSA to the column. Push the column plunger to collect the CD31-positive fraction. Centrifuge the sample as demonstrated earlier and resuspend the cell pellet in one milliliter of complete microvascular endothelial cell growth media.Dispense the suspension into two wells of a 2%gelatin-coated 24-well plate. Culture the cells at 37 degrees Celsius with 5%carbon dioxide for two to four weeks until cells become nearly 80%confluent.