Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Summary

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Abstract

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

Introduction

Calcific aortic valve disease (CAVD) is a chronic pathology characterized by inflammation, fibrosis, and macrocalcification of aortic valve leaflets. Progressive remodeling and calcification of the leaflets (termed aortic sclerosis) can lead to the obstruction of blood flow (aortic stenosis) which contributes to stroke and leads to heart failure. Currently the only treatment for CAVD is surgical or transcatheter aortic valve replacement (SAVR and TAVR, respectively). There is no non-surgical option to halt or reverse CAVD progression, and without valve replacement, mortality rates approach 50% within 2-3 years^1,2,3. Defining the underlying mechanisms driving this pathology will identify potential novel therapeutic approaches.

In a healthy adult, aortic valve leaflets are approximately one millimeter thick, and their main function is to maintain the unidirectional flow of blood out of the left ventricle⁴. Each of the three leaflets is comprised of a layer of valve endothelial cells (VECs) that lines the outer surface of the leaflet and functions as a barrier. VECs maintain valve homeostasis by regulating permeability, inflammatory cell adhesion, and paracrine signaling^5,6,7. Valve interstitial cells (VICs) comprise the majority of cells within the valve leaflet⁸. VICs are arranged in three distinctive layers in the leaflet. These layers are known as the ventricularis, the spongiosa, and the fibrosa⁹. The ventricularis faces the left ventricle and contains collagen and elastin fibers. The middle layer, the spongiosa, contains high proteoglycan content that provides shear flexibility during the cardiac cycle. The outer fibrosa layer is located close to the outflow surface on the aortic side and is rich in Type I and Type III fibrillar collagen which provide strength to maintain coaptation during diastole^10,11,12. VICs reside in a quiescent state, however, factors such as inflammation, remodeling of the extracellular matrix (ECM), and mechanical stress may disrupt VIC homeostasis^{8,9,13,14,15,16}. With loss of homeostasis, VICs activate and acquire a myofibroblast-like phenotype capable of proliferation, contraction, and secretion of proteins that remodel the extracellular millieu¹⁷. Activated VICs can transition into calcifying cells which is reminiscent of the differentiation of a mesenchymal stem cell (MSC) into an osteoblast^{15,17,18,19,20,21,22,23,24},²⁵.

Calcification appears to initiate in the collagen-rich fibrosa layer from contributions of both VECs and VICs but expands and invades the other layers of the leaflet⁸. Thus, it is clear that both VECs and VICs respond to stimuli to upregulate the expression of osteogenic genes, however, the precise events driving the activation of osteogenic genes, as well as the complex interplay between the cells and the extracellular matrix of the leaflet, remain ill-defined. Murine models are not an ideal source to study non-genetic drivers of CAVD pathogenesis, as mice do not develop CAVD de novo^26,27, hence the use of primary human tissues and the primary cell lines isolated from these tissues is necessary. In particular, obtaining these cells in high numbers and good quality is imperative, as the field of 3D cell cultures and organoid modeling is expanding and is likely to become an ex vivo human-based alternative to murine models.

The purpose of the present method is to share a workflow that has established the conditions to efficiently isolate and grow VECs and VICs obtained from surgically removed valves from human donors. Previous studies have shown successful isolation of VECs and VICs from porcine²⁸ and murine valves²⁹, to our knowledge this is the first to describe the isolation of these cells in human tissues. The protocol described here is applicable to human excised valves and greatly circumvents and improves the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing by introducing the utilization of a cold storage solution, a buffered solution clinically utilized in organ transplants that greatly stabilizes cells of the excised tissue. The protocol described here also shows how to determine cell phenotype and guarantee high efficiency of cell survival with minimal cell cross-contamination.

Protocol

All patient samples are collected from individuals enrolled in studies approved by the institutional review board of the University of Pittsburgh in accordance with the Declaration of Helsinki. Cadaveric tissues obtained via the Center for Organ Recovery and Education (CORE) were approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents (CORID).

1. Approval and safety

1. Obtain Institutional Review Board (IRB) approval or an exempt memo for any collection of patient samples or cadaveric tissues in accordance with the Declaration of Helsinki.
2. Take required institutional training to work with human tissues such as Bloodborne Pathogens Training, Biomedical Human Subjects Research, Privacy and Information Security, and Transportation and Shipping of Biological Materials.

2. Logistics and preparation

1. Surgical Samples
   1. Ensure that a refrigerator is available and located near the operating room. Keep 50 mL conical tubes pre-labeled with de-identified nomenclature containing 40 mL of sterile aliquots of cold storage solution in this fridge for use by surgical staff. These tubes are stable at 4 °C until expiration date on the original package.
   2. Upon extraction, place valve tissue in these tubes. Place the tubes in a sealed secondary container such as a leak-proof plastic bag or a plastic container that is labeled with a biohazard label. Tissues are picked up and transported on ice according to institutional protocols for transporting biohazards.
2. Cadaveric samples
   1. Submerge recovered organs in cold storage solution, place in a sealed secondary containment vessel, and transport on ice according to institutional protocols for transporting biohazards.

3. Reagents preparation

1. Make collagen-coated plates at least the day before.
   1. In a 50 mL conical tube, mix 5 mL of isopropyl alcohol, 8.7 mL of acetic acid, and 0.5 µg of collagen I powder. Bring up to 50 mL with sterile water. Mix and filter through a 0.45 µm filter.
   2. Under a sterile cell culture hood, add enough collagen solution to 6 well plates and 10 cm dishes to just cover the entire bottom. Cover the plate and let sit for 4-6 h at room temperature. Remove the excess solution with a sterile pipette, place in a new sterile 50 mL conical tube, and save at 4 °C to make additional plates. Solution can be stored at 4 °C for several months.
   3. Dry the plates in a 37 °C incubator overnight, and subsequently store them in a resealable bag at 4 °C. Collagen-coated plates and dishes can be stored for several months.
2. Autoclave the following items: tissue forceps, tissue scissors, cotton swabs and gauze pads.
3. VEC growth media: Prepare and use endothelial cell growth medium according to manufacturer's protocols. Store in the dark at 4 °C. Warm to 37 °C just before use on cells, do not leave media in warming bath longer than necessary (10-15 min is sufficient). Use the media within one month of preparation.
4. VIC growth media: Supplement DMEM base medium (4.5 g/L glucose, L-glutamine supplement, 110 mg/L sodium pyruvate)³⁰ with 10% heat-inactivated FBS and 100 I.U./mL penicillin and 100 μg/mL streptomycin. Store in the dark at 4 °C for up to 3 months. Warm to 37 °C just before use on cells, do not leave media in warming bath longer than necessary (10-15 min is sufficient).
5. Make sterile rinsing solution just before use. Supplement sterile PBS with 2.5 µg/mL fungicide, 0.05 mg/mL gentamicin and 5 µg/mL bactericide.
6. Make sterile collagenase solution just before use. Add 5 mg of collagenase II to 5 mL of sterile fresh DMEM base medium. Mix well and sterilize the solution by passing through a 0.45 µm filter. Keep on ice until use.

4. Tissue preparation and processing

NOTE: Institutional approval for use of human tissues must be obtained prior to beginning work. While handling tissues, the following personal protective equipment (PPE) must be worn: a disposable liquid barrier wrap-around gown, or a dedicated button front lab coat with a liquid-barrier wrap around apron and disposable sleeve clovers; a full face shield, or safety glasses with a surgical mask; double gloves; close-toed shoes; and clothing to cover the legs. Comprehensive workflow diagrams of the tissue preparation for calcification assessment (Section 5) and cell isolation (Sections 6 and 7) are illustrated in Figure 1A,B respectively.

1. Upon receipt of cadaveric organ specimen, excise the aortic root and submerge in a 50 mL conical tube of sterile rinsing solution. Upon receipt of surgical specimen, remove from transport vessels and submerge in a 50 mL conical tube of sterile rinsing solution. Place tube containing the tissue in ice bucket on rocker and mix for 10 min at room temperature (RT).
   NOTE: Processing tissues as close as possible to the time of extraction will yield the best cell recovery, however viable cells can be collected upwards of 61 h post excision, and data show that VICs are more robust than VECs as time increases. If tissue cannot be processed immediately, when possible, remove tissue, perform step 4.1, and then put the tissue back in fresh sterile cold storage solution and keep at 4 °C until ready to proceed. Valves collected during night or weekend surgeries can be stored in 40 mL cold storage solution at 4 °C and still are able to yield viable cells more than 2 days post extraction.
2. Spray down tubes with 70% ethanol and move to a sterile hood. Remove tissue and excise two valve leaflets (Figure 2A). Place one leaflet in a cryogenic vial (or 2-3 vials if several smaller pieces are needed for future analysis) and snap freeze by dropping in liquid nitrogen then store at -80 °C.
   1. If the valve is bicuspid, excise only one leaflet. Using scissors, cut the leaflet in half from the nodule to the hinge. Use one half of the leaflet for snap freezing and the other half for step 4.3.
3. Process the second leaflet for paraffin embedding to assess calcification content by cutting it in half from nodule to hinge. Place both pieces in a cassette that is submerged in 4% paraformaldehyde (PFA), then place on a rocker at RT for a minimum of 2 h but no more than 4 h.
   NOTE: Longer fixation times create more background with immunofluorescent staining. Once steps 4.2 and 4.3 are performed, move immediately to Section 6 and come back to step 4.4 after step 6.12.
4. After fixation, wash the tissues by submerging in fresh PBS for 1 h 3-4 times. After these washes, samples can stay in PBS at 4 °C for several months if needed. Proceed to next step just before embedding.
5. Gradually change from PBS to 70% ethanol. Wash 30-60 min each step with 1:4 70% ethanol:PBS; 1:1 70% ethanol:PBS, 4:1 70% ethanol:PBS; 70% ethanol.
6. Embed the tissue such that sections will reveal the three layers of the leaflet (Figure 1A) according to established protocols³¹. Alternatively, and if available, bring the tissue to a pathology core for embedding and cutting.
7. After handling tissues, dispose or store PPE as appropriate and wash hands immediately. Decontaminate all equipment, surfaces, and solid and liquid wastes with a 1:10 dilution of bleach or detergent disinfectant. Allow 20 min for decontamination, then follow with a 70% ethanol rinse. Treat the cell culture hood with mycoplasma spray according to manufacturer's instructions.

5. Von Kossa staining for calcium content

NOTE: This can be done well after cell isolation and line establishment but be sure to link the calcification level of the tissue to documents pertaining to the primary cell line established.

1. Cut 5 or 10 µm thick paraffin slices onto glass slides and bake slides at 65 °C for 1 h, then cool to RT.
2. Using fresh solutions, deparaffinize slides by submerging as following: 100% xylene for 30 min, 2x; 100% ethanol for 3 min, 2x; 90% ethanol for 3 min; 80% ethanol for 3 min; 70% ethanol for 3 min; 50% ethanol for 3 min; ultrapure water for 3 min; keep in ultrapure water until next steps.
   NOTE: Times above are minimum, each step may go longer.
3. Proceed with Von Kossa staining according to manufacturer's protocols.
4. Assess full valve area microscopically and assign a control tissue (no sign of calcification) or CAVD (any evidence of calcification, Figure 2B).

6. Valve Endothelial Cell (VEC) isolation, expansion, and confirmation

1. In a sterile cell culture hood, open the conical tube containing the remaining valve leaflet and place the leaflet in a new 50 mL conical tube filled with ice cold PBS. Cap tube and gently invert or place on a rocker for 2 min at RT.
2. Remove tissue to a 60 mm dish filled with 5-7 mL of cold collagenase solution. Using forceps, dip both sides of the leaflet in the solution 3-4 times. Incubate the tissue for 5-10 min in the cell culture incubator at 37 °C, rocking the tissue gently every 2 min 3-4 times.
3. Remove 2 mL of the solution from the dish and place in a sterile 15 mL conical tube. Place forceps at the nodule and use a dry sterile cotton swab to swipe from the forceps to the hinge, twirling the swab while moving it along the leaflet. Between each swipe, swish the swab in the solution in the 15 mL conical tube to remove the cells. Repeat to fully swab the surface of the tissue, then flip over and repeat on the other side.
4. Holding the valve leaflet with forceps in one hand, and a 1 mL pipette in the other, rinse the surfaces of the leaflet with the solution in the dish. Once rinsed, transfer all the solution containing the VECs in the dish into the same 15 mL conical tube with the cells from the swab and proceed to Step 6.5. Place the remaining valve tissue in a new 15 mL conical tube with 7 mL of sterile collagenase solution and proceed to Step 7.1.
5. Centrifuge the tube containing the VECs at 180 x g for 5 min to pellet the isolated VECs. Aspirate the supernatant and resuspend in 3 mL of VEC growth media. Centrifuge one more time and remove the supernatant. Resuspend the cells in 1 mL of growth media and determine the number of cells using a hemocytometer and trypan blue.
6. Resuspend VEC pellet in 2 mL of VEC growth media and plate the cells in a collagen pre-coated well of a 6 well plate at approximately 5 x 10⁵ cells/cm². Let the cells grow at least 3-4 days, then remove media and replenish with fresh media. Repeat media changing every 4 days. VECs will grow in cobblestone shaped patches (Figure 3B, left panels).
7. When VEC patches cover >80% of the plate, split cells at about 1.3 x 10⁴ cells/cm² depending on how fast they grow (if they reach 80% in less than 1 week split at a slightly lower number of cells/cm²).
8. To split, wash cells two times with 2 mL DPBS then add enough dissociation reagent to just cover the surface of the cells. Incubate for 2-3 min at 37 °C, checking to make sure cells are not over incubated. Stop digestion by adding equal volume of VEC growth media and transfer liquid to a 15 mL tube. Centrifuge at 180 x g for 5 min, remove supernatant, and resuspend in appropriate volume of media for number of wells/plates needed.
   NOTE: Once expanded into a 10 cm plate VECs may sometimes lose their morphology and change phenotype as they proliferate. This tends occurs when VECs are seeded at a low confluency during the establishment and expansion of the cell line.
9. To guarantee a pure culture consider utilizing CD31+ superparamagnetic beads with every splitting.
10. For 1 x 10⁸ or fewer cells (one 10 cm dish or less), prepare 25 µL of superparamagnetic beads by washing according to manufacturer's protocols.
    NOTE: Step 6.10 is recommended to be performed before trypsinization of VECs.
11. Detach cells as in step 6.8 but resuspend in 500 µL of PBS with 0.1% BSA, pH 7.4, and place in a 2 mL centrifuge tube. Add 500 µL of washed and resuspended beads to the 2 mL tube of cells and incubate on a rotator for 20 min at 4 °C.
12. Place tubes on the magnet for 2 min. While tube is still in the magnet, carefully remove supernatant. Remove tube from magnet, add 1 mL fresh PBS with 0.1% BSA, pipette gently 2-3 times, then place back on magnet for 2 min. Repeat 2 more times. After final removal of buffer, resuspend cells in growth media in volume needed for replating.
    NOTE: Cells will still have beads, but these will not affect growth and will be removed in subsequent passages.
13. Once cells have been expanded, in addition to morphology, confirm VEC phenotype by positive staining for immunofluorescent markers such as von Willebrand factor (vWF), cadherin 5 (CDH5), or PECAM-1/CD31, and negative staining for VIC markers such as calponin 1 (CNN) or alpha-2 smooth muscle actin (αSMA, Figure 4, left panels).

7. Valve Interstitial Cell (VIC) isolation, expansion, and storage

1. As stated in step 6.4, after removing VECs from the valve tissue, the leaflet is placed in a 15 mL conical tube with 7 mL collagenase solution. Incubate for 12 h in the cell culture incubator with the cap slightly open to allow gas exchange. Successful isolation of VICs can still occur with up to 18 h in collagenase solution.
2. After incubation, in a sterile cell culture hood, mix the tissue gently by pipetting with a serological pipette to ensure the release of VICs from the leaflet tissue.
3. Remove cell suspension and pass through a 0.70 μm filter into a 50 mL conical tube.
4. Add 7 mL of VIC growth medium to the 50 mL tube and centrifuge at 180 x g for 5 min. Aspirate supernatant and resuspend cell pellet in 1 mL of VIC growth media and determine the cell number. Plate the VICs in a 60 mm tissue-culture treated dish at 1.3 x 10⁴ cells/cm².
5. Let the cells grow at least 1-2 days before replacing media. Remove media and wash twice DPBS to remove residual debris and replace with fresh growth medium. Replenish medium every 2-3 days. VICs will grow in fibroblast shape (Figure 3B, right panels).
6. When VICs reach a confluency of >90%, wash twice with DPBS to remove the excess media and detach the cells by adding appropriate volume of pre-warmed dissociation reagent to cover the plate (i.e., 2-3 mL per 10 cm dish). Incubate the dish in a 37 °C incubator. VICs will detach from the dish after 2-3 min of incubation. Add 4-6 mL of pre-warmed media.
   NOTE: If cells take longer than 3 min to lift off the plate, the dissociation reagent may have lost potency. If needed, a cell scraper may be used to gently lift the cells.
7. Transfer the cell suspension to a tube and gently centrifuge at 180 x g for 5 min. After removing the supernatant, gently resuspend the cell pellet in pre-warmed VIC growth media and determine the number of viable cells by using a hemocytometer and trypan blue. Seed the viable cells at a 1:2 density of the original dish (i.e., ~ 1 × 10⁶ cells per 10 cm dish or 1.3 x 10⁴ cells/cm²).
8. Assess VIC phenotype by positive staining for immunofluorescent markers such as αSMA, CNN, or SM22α and negative staining for VEC markers such as CD31, CDH5, or vWF (Figure 4, right panels).
   NOTE: The protocol workflow presented here unbiasedly selects one leaflet for VECs and VICs isolation, while the remaining leaflets are utilized for Von Kossa staining (Section 5) and snap freezing.

8. Long-term cell storage

1. Once expanded, freeze cells down for long-term storage. Wash cells twice with 2-5 mL DPBS then add enough dissociation reagent to detach cells as in steps 6.8 and 7.6. Stop digestion by adding equal volume of VEC or VIC growth media and transfer cell suspension to a 15 mL tube.
2. Determine the number of viable cells by using a hemocytometer and trypan blue.
3. Centrifuge at 180 x g for 5 min.
4. Resuspend cells in chilled conditioned growth media to a cell density of ~ 3 × 10⁶ cells/mL.
5. Gently with swirling, add an equal volume of chilled 2x cryopreservation medium. This will bring cell concentration to ~ 1.5 × 10⁶ cells/mL.
6. Aliquot 1 mL into cryopreservation vials. Place vials in a cell freezing container, close, and invert 5-6 times to ensure cells remain suspended. Place container at -80 °C for 6-72 h or according to freezing container protocol. Remove vials from -80 °C and transfer to liquid nitrogen for long term storage.

Results

The above protocol outlines the steps necessary for the handling of human valve tissues and the isolation and establishment of viable cell lines from these tissues. Leaflets of the aortic valve are processed for paraffin embedding, snap frozen for long term storage for biochemical or genetic analysis and digested for the isolation of VECs and VICs (Figure 1). While surgical specimens will likely have a clinical diagnosis of aortic stenosis and may exhibit heavy nodules of calcification that ...

Discussion

Obtaining control and disease tissues from humans is critical for in vitro and ex vivo disease modeling; however, while one often speaks about the challenges of bridging the gap between bench to bedside, the reverse order - going from the surgical suite to the bench - is often just as daunting a gap. Essential for a basic scientist to obtain primary human tissue specimens is a collaboration with an invested surgeon scientist who has a team of nurses, surgical technicians, physician assistants, medical students and reside...

Materials

Name	Company	Catalog Number	Comments
0.45 μm filter	Thermo Scientific	7211345	Preparing plate with collagen coating
10 cm cell culture plate	Greiner Bio-One	664160	Cell culture/cell line expansion
10 mL serological pipet	Fisher	14955234	VEC/VIC isolation, cell culture, cell line expansion
1000 μL filter tips	VWR	76322-154	Cell culture/cell line expansion
10XL filter tips	VWR	76322-132	Cell culture/cell line expansion
15 mL conical tubes	Thermo Scientific	339650	Tissue storage, VIC/VEC isolation
16% paraformaldehyde aqueous solution	Electron Microscopy Sciences	15710S	Tissue and cell fixative
190 proof ethanol	Decon	2801	Disinfection
1x DPBS: no calcium, no magnesium	Gibco	14190250	Saline solution. VIC/VEC isolation
1x PBS	Fisher	BP2944100	Saline solution. Tissue preparation, VIC/VEC isolation
20 μL filter tips	VWR	76322-134	Cell culture/cell line expansion
200 proof ethanol	Decon	2701	Deparaffinizing tissue samples
2-propanol	Fisher	A416P 4	Making collagen coated plates
5 mL serological pipet	Fisher	14955233	VEC/VIC isolation, cell culture, cell line expansion
50 mL conical tubes	Thermo Scientific	339652	Tissue storage, VIC/VEC isolation
60 mm dish	GenClone	25-260	VEC isolation
6-well cell culture plate	Corning	3516	Cell culture/cell line expansion
Acetic acid, glacial	Fisher	BP2401 500	Making collagen coated plates
AlexaFluor 488 phalloidin	Invitrogen	A12379	Fluorescent f-actin counterstain
Belzer UW Cold Storage Transplant Solution	Bridge to Life	BUW0011L	Tissue storage solution
Bovine Serum Albumin, Fraction V - Fatty Acid Free 25g	Bioworld	220700233	VEC confirmation with CD31+ Dynabeads
Calponin 1 antibody	Abcam	ab46794	Primary antibody (VIC positive stain)
CD31 (PECAM-1) (89C2)	Cell Signaling	3528	Primary antibody (VEC positive stain)
CD31+ Dynabeads	Invitrogen	11155D	VEC confirmation with CD31+ Dynabeads
CDH5	Cell Signaling	2500	Primary antibody (VEC positive stain)
Cell strainer with 0.70 μm pores	Corning	431751	VIC isolation
Collagen 1, rat tail protein	Gibco	A1048301	Making collagen coated plates
Collagenase II	Worthington Biochemical Corporation	LS004176	Tissue digestion. Tissue preparation, VIC/VEC isolation
Conflikt Ready-to-use Disinfectant Spray	Decon	4101	Disinfection
Countess II Automated Cell Counter	Invitrogen	A27977	Automated cell counter
Countess II reusable slide coverslips	Invitrogen	2026h	Automated cell counter required slide cover
Coverslips	Fisher	125485E	Mounting valve samples
Cryogenic vials	Olympus Plastics	24-202	Freezing cells/tissue samples
Disinfecting Bleach with CLOROMAX - Concentrated Formula	Clorox	N/A	Disinfection
DMEM	Gibco	10569044	Growth media. VIC expansion
EBM - Endothelial Cell Medium, Basal Medium, Phenol Red free 500	Lonza Walkersville	CC3129	Growth media. VEC expansion
EGM-2 Endothelial Cell Medium-2 - 1 kit SingleQuot Kit	Lonza Walkersville	CC4176	Growth media supplement. VEC expansion
EVOS FL Microscope	Life Technologies	Model Number: AME3300	Fluorescent imaging
EVOS XL Microscope	Life Technologies	AMEX1000	Visualizing cells during cell line expansion
Fetal Bovine Serum - Premium Select	R&D Systems	S11550	VIC expansion
Fine scissors	Fine Science Tools	14088-10	Tissue preparation, VIC/VEC isolation
Fisherbrand Cell Scrapers	Fisher	08-100-241	VIC expansion
Fungizone	Gibco	15290-026	Antifungal: Tissue preparation, VIC/VEC isolation
Gentamicin	Gibco	15710-064	Antibiotic: Tissue preparation, VIC/VEC isolation
Glass slides	Globe Scientific Inc	1358L	mounting valve samples
Goat anti-Mouse 488	Invitrogen	A11001	Fluorescent secondary Antibody
Goat anti-Mouse 594	Invitrogen	A11005	Fluorescent secondary Antibody
Goat anti-Rabbit 488	Invitrogen	A11008	Fluorescent secondary Antibody
Goat anti-Rabbit 594	Invitrogen	A11012	Fluorescent secondary Antibody
Invitrogen Countess II FL Reusable Slide	Invitrogen	A25750	Automated cell counter required slide
Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (DAPI)	Invitrogen	R37606	Fluorescent nucleus counterstain
LM-HyCryo-STEM - 2X Cryopreservation media for stem cells	HyClone Laboratories, Inc.	SR30002	Frozen cell storage
Mounting Medium	Fisher Chemical Permount	SP15-100	Mounting valve samples
Mr. Frosty freezing container	Nalgene	51000001	Container for controlled sample freezing
Mycoplasma-ExS Spray	PromoCell	PK-CC91-5051	Disinfection
Penicillin-Streptomycin	Gibco	15140163	Antibiotic. VIC expansion
Plasmocin	Invivogen	ANTMPT	Anti-mycoplasma. VIC/VEC isolation and expansion
SM22a antibody	Abcam	ab14106	Primary antibody (VIC positive stain)
Sstandard pattern scissors	Fine Science Tools	14001-14	Tissue preparation, VIC/VEC isolation
Sterile cotton swab	Puritan	25806 10WC	VEC isolation
Swingsette human tissue cassette	Simport Scientific	M515-2	Tissue embedding container
Taylor Forceps (17cm)	Fine Science Tools	11016-17	Tissue preparation, VIC/VEC isolation
Trypan Blue Solution, 0.4%	Gibco	15250061	cell counting solution
TrypLE Express Enzyme	Gibco	12604021	Splitting VIC/VECs
Von Kossa kit	Polysciences	246331	Staining paraffin sections of tissues for calcification
von Willebrand factor antibody	Abcam	ab68545	Primary antibody (VEC positive stain)
Xylenes	Fisher Chemical	X3S-4	Deparaffinizing tissue samples
αSMA antibody	Abcam	ab7817	Primary antibody (VIC positive stain)