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.
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.
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 years1,2,3. 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 signaling5,6,7. 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 diastole10,11,12. VICs reside in a quiescent state, however, factors such as inflammation, remodeling of the extracellular matrix (ECM), and mechanical stress may disrupt VIC homeostasis8,9,13,14,15,16. 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 osteoblast15,17,18,19,20,21,22,23,24,25.
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 novo26,27, 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.
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
2. Logistics and preparation
3. Reagents preparation
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.
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.
6. Valve Endothelial Cell (VEC) isolation, expansion, and confirmation
7. Valve Interstitial Cell (VIC) isolation, expansion, and storage
8. Long-term cell storage
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 can be visible with the naked eye, aortic valve calcification is present in a significant number of elderly (>65 years old) individuals32, and because of this prevalence all tissues – surgical and cadaveric – are subjected to Von Kossa staining or similar procedure to assess whether calcification is present (Figure 2).
It was found that use of cold storage solution greatly stabilized the cells of the excised valve tissue. Cold storage solution is used in live organ transplants. It is flushed through organs either before or after removal from the donor and left in the vasculature of that organ during transportation on ice. It was observed that VEC lines were more readily established from donor specimens than surgical tissues, and the donor lines were more likely to retain their endothelial cell morphology for more passages. This was perplexing, as donor tissues would often not reach the lab for 12-24 hours postmortem while surgical tissue was obtained between 2 and 4 hours. Upon packing the surgical specimen tubes with cold storage solution instead of PBS, cell recovery greatly increased. As seen in Figure 3, both viable VECs and VICs can be obtained upwards of 61 hours post valve extraction. While a slightly higher percentage of live VICs are obtained than VECs when cells are isolated up to a day after valve excision, cells remain viable after 48 hours post excision. Up to 40% of the total recovered cells correspond to alive cells and we have observed consistent figures between biological replicates. Further, morphology inspection also confirms cell identity; while VECs appear as packed, cobblestone-like and growth contact-inhibited cells, VIC morphology is similar to myofibroblasts with a spindle shape. Immunostaining of the specimens confirmed that 91.8% ± 1.8 (n = 3) of expanded VECs were positive for the endothelial marker vWF whereas 92.0% ± 5.0 (n = 3) of expanded VICs were positive for the interstitial cell marker αSMA (Figure 4). These figures are in line with results reported previously33.
Figure 1: Valve tissue processing and cell isolation from human control and CAVD valves. (A) Schematic representation of tissue processing for assessment of calcification levels of the valves. (B) Schematic representation of the time course and steps for the isolation and characterization of human valve endothelial cells (VECs) and valve interstitial cells (VICs) from control and CAVD tissues. Please click here to view a larger version of this figure.
Figure 2: Assessment of Calcification. (A) Representative image of control (top) and calcified (bottom) valve tissues from human donors. Note the calcification nodules of the CAVD tissue can severely alter morphology and the ability to cleanly excise the leaflets. (B) Representative Von Kossa staining of control (left) and CAVD (right) valve tissue after fixation and further processing of the human valve tissue. Note calcification is revealed by the presence of dark precipitation in the leaflet tissue. Valve images were capture with a lab camera. Von Kossa stained valves were captured with a 10x objective, scale bar 200 µm. Please click here to view a larger version of this figure.
Figure 3: Assessment of Cell Survival (A) VECs and VICs survival curves. Three leaflets were obtained from five valve specimens. The first leaflet from each valve was processed immediately (3-12 h post extraction), the second leaflet was processed approximately 24 h later (22-35 h), and the third leaflet was processed approximately 48 h after obtaining the tissue (45-61 h). Valves leaflets were kept in cold storage solution at 4 °C until they were processed. The y-axis represents the percentage of live cells. (B) Morphology of healthy cultures of VECs (left panels) and VICs (right panels). Graphs show mean ± SD live cell proportion of cells isolated from n = 5 valve tissues. Images were captured with a 4x and 10x objective, scale bars 200 µm and 100 µm, respectively. Please click here to view a larger version of this figure.
Figure 4: Representative immunofluorescent staining on VECs and VICs. VECs are positive for the endothelial marker von Willebrand Factor (vWF, left panels) whereas VICs are positive for the interstitial marker αSMA. Note our isolation protocol guarantees a high isolation efficiency; no cross-contamination between VECs and VICs is detected. Images were captured with a 10x objective, scale bar 100 µm. Please click here to view a larger version of this figure.
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 residents, and clinical protocol managers who can enroll and consent patients, participate and assist in the proper handling of excised tissues, and coordinate the logistics required for tissue pickup. Without the utmost effort from everyone involved to reduce the time from excision to cell isolation, vital cellular material and the information it contains will be irreparably altered or lost.
Critical to maintaining viability of the tissue specimens is the use of cold storage solution. This is the same solution used by the organ transplant teams at UPMC and other transplant medical centers. Better cell yield was obtained from cadaveric tissues that had been excised from the donor many hours beforehand than from tissues obtained more quickly from the operating room but kept in cold PBS. This accidental discovery has been essential for cell procurement from human tissues. Procurement time from tissue excision to delivery in the laboratory ranges from 1-5 hours for surgical tissue and upwards of 24 hours for cadaveric tissue. In comparison, procurement and processing of animal tissues can often be done within minutes of euthanasia, which is ideal for cell viability. In the absence of cold storage solution, it is likely that a medium suitable for the culturing of cells could also perform better than PBS, however this medium was not tested herein due to the success of the cold storage solution in live organ transplantation and to the receipt of cadaveric tissues in this solution. The solution is shelf-stable which is ideal for storage in operating rooms, and specific ingredients such as adenosine are known to promote beneficial responses to cellular stresses such as ischemia/hypoxia21,34.
Another essential step to obtaining viable cells is washing the tissues with fungicide, gentamicin, and bactericide. This short rinse helps to ensure that cells remain uncontaminated by bacteria and fungus. Equally critical are the steps to digest VECs out of the valve tissue, where in the span of just a few minutes, the VECs are detached and then swabbed off the surface of the valve leaflets. The subsequent digestion of the VICs that reside on the dense extracellular matrix of the leaflet has much more wiggle room for duration and strength. The unbiased tissue treatment described in this protocol allows the isolation of the main two cell populations present in the valve leaflet, VECs and VICs. Although a recent single cell transcriptome analysis has shown the co-existence of at least fourteen different cell subtypes residing in the human valve, including six non-valve derived stromal cells in CAVD tissue35, this diversity may represent variations due to effects from processing and digesting these hardened tissues, or it may be due to different microenvironments to which the leaflet cells are exposed: VECs are exposed to two different blood flows while VICs are embedded in three different extracellular matrix stratums8,9. The large-scale isolation protocol and analysis described herein ensure that over 90% of VECs and VICs correspond to their main phenotype. Although a degree of heterogenicity may be found, it does not affect the general outcomes of the study of VEC and VIC homeostasis8,9,13,14,15,16.
It is also important to note that patients and their valve tissues, and thus the cell lines procured from them, are not identical. Genetics, co-morbidities, handling during surgery and processing, and freshness of digestion solution ingredients may affect the growth rate and even perhaps the behavior or phenotype of the cells isolated. While the present study demonstrates the ability to yield a sufficient number of viable cells with this procedure, there may be innate or induced differences in these cell lines that may impact downstream experimentation. It is often difficult to know precisely the time since the tissue has been removed from the patient or donor, and particularly in the case of the latter, the time since circulation has stopped. Further, there is inherent variability among tissues regarding the number of viable valve cells obtained, the proliferative capacity of the cells, and the retention of cell phenotype. Cell lines may harbor genetic mutations – either congenital or somatic – of which the physician and research team are unaware, and the remodeling and subsequent handling of the tissues may also modify the cell phenotype or even epigenetics. As such, for all experiments in which these primary human cells are used, it is absolutely essential that biological replicates – i.e., cell lines obtained from different patients – be used, despite the substantial time and cost they incur. This helps ensure that any results are not due to confounding effects from the procurement and processing of the tissue. The variable proliferation rate of different cell lines can be adjusted for by either collecting cells in experiments at different times or seeding cells for experiments at different densities; no one answer is best for all experimental designs. While the complications are not insignificant, the use of primary human control and CAVD tissue-derived cell lines for in vitro and ex vivo experimental models is essential for defining the initiating factors and propagating processes that drive CAVD pathogenesis.
The authors have nothing to disclose.
We would like to thank Jason Dobbins for insightful discussion and critical reading of this manuscript. We would like to acknowledge the Center for Organ Recovery and Education for their help and support and thank tissue donors and their families for making this study possible. 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).
Some figures created with Biorender.com.
CSH is supported by the National Heart, Lung, and Blood Institute K22 HL117917 and R01 HL142932, the American Heart Association 20IPA35260111.
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) |