fixing and blocking 2d human umbilical vein endothelial cell (huvec) cultures for immunohistochemical analysis

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Angiogenesis, which refers to the formation of new blood vessels from pre-existing ones1, is a crucial process during physiological development and pathologic conditions. It is indispensable for providing energy to highly metabolically active tissues such as the retina2 or the developing central nervous system3 and during the healing of damaged tissue4. Abnormal angiogenesis, on the other hand, is the basis for numerous diseases. Solid tumors, such as colorectal cancer or non-small cell lung cancer, facilitate their growth and the necessary energy supply by promoting angiogenesis5. Apart from cancer, neovascular diseases of the eye like diabetic retinopathy or age-related macular degeneration, which represent leading causes of blindness in the developed world, result from aberrant vessel growth6,7. A detailed understanding of the underlying pathomechanism is crucial to comprehend how physiological angiogenesis can be enhanced, for instance, in wound healing while better controlling pathological conditions such as vasoproliferative eye diseases.
On a cellular level, vascular endothelial cells are activated by various signaling molecules in angiogenesis, initiating cell proliferation and migration8. The cells subsequently organize into a hierarchy, with non-proliferating tip cells forming filopodia at the leading edge of the developing vessel branch8. Alongside, fast-proliferating stalk cells trail the tip cells, contributing to the formation of the emerging vessel. Subsequently, other cell types, such as pericytes or smooth muscle cells, are recruited to further stabilize the nascent branch9.
To explore molecular processes at the vascular endothelial cell level, numerous in vitro protocols have been developed and recently reviewed10. These assays typically fall into two categories: more simplistic but scalable 2D approaches and more elaborate 3D protocols. In a recent project, we conducted a comprehensive comparative analysis between the 2D scratch wound migration assay and the 3D spheroid sprouting assay11 to assess the extent of their differences and their ability to model various aspects of angiogenesis12. 
While both offer the advantages of being reliable and easily implementable, on a molecular level, the 3D spheroid sprouting assay was favorable in addressing key aspects of angiogenesis compared to in vivo data, such as metabolic switches or cell-matrix interactions. Since in vitro angiogenesis assays are used to evaluate the angiomodulatory potential of signaling pathways13 and to screen for therapeutic agents, transferability of in vitro results to in vivo settings is essential. Furthermore, the opportunity for omics-based analyses on the RNA and protein levels to characterize the molecular changes in response to targeted modulation of angiogenic processes under controlled conditions remains an important benefit compared to in vivo settings14,15.
In this publication, we present key assays for exploring angiogenesis-related questions through the utilization of a live-cell imaging migration assay and a spheroid sprouting assay, including subsequent molecular analyses like RNA sequencing for transcriptomic analysis and immunohistochemistry on the protein level.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis is a hallmark process involved in embryology and in disease development, including tumor growth and vasoproliferative eye disease. In this project, we aim to present a comprehensive framework of in vitro techniques that can be used to study angiogenesis on a molecular level in a very controlled setting, and to screen for potential therapeutic options. Translating in vitro to in vivo and ultimately to the clinical setting is challenging due to the risk of false positive or false negative results.Selecting the right angiogenesis assay is therefore crucial and knowing its limitations. This study aims to offer guidelines in how to interpret and how to establish two common angiogenesis assays. Recently, we compared a 2D scratch wound migration assay with a 3D spheroid sprouting assay.While the 2D assay is easily scalable, the 3D assay captures angiogenesis in greater detail, including tip stalk cell formation, matrix interaction, and the glycolytic switch. This data offer a scientists guidelines on which assay to choose next in their projects. Our laboratory will continue to characterize key aspects of the presented in vitro assays by directly comparing them to in vivo settings in order to facilitate translations of in vitro results to clinical applications.

Fixing and Blocking 2D Human Umbilical Vein Endothelial Cell (HUVEC) Cultures for Immunohistochemical Analysis

To begin, add 30 milliliters of 5%potassium hydroxide in methanol to a 50 milliliter tube. Incubate 100 cover slips with a diameter of 12 millimeters in the tube for 30 minutes under constant agitation. Next, wash the cover slips in demineralized water for 30 minutes.Once washing is complete, transfer the cover slips into 70%isopropyl solution for storage. To coat the cover slips, first lean them individually against the wall of a square Petri dish. When the isopropyl alcohol has evaporated, transfer them into a 24 well plate.Pipette one milliliter of collagen in PBS into each well and incubate. Next, wash the cover slips about 4 to 5 times for five minutes each with one milliliter of PBS. Pipette cultured HUVECs into the wells with the coated cover slips.After culturing the cells for a desired length of time, wash the cells in one milliliter of PBS for five minutes. Fix the cells in one milliliter of 2%paraformaldehyde for 20 minutes at room temperature. Now, rinse the cells in one milliliter of PBS for 3 to 4 wash cycles of five minutes each.Finally, pipette one milliliter of blocking buffer into the well plate before staining.

fixing-blocking-2d-human-umbilical-vein-endothelial-cell-huvec

Research

JoVE Journal

Medicine

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

Scratch Wound Migration Assay of Human Umbilical Vein Endothelial Cells (HUVECs) for Investigating Angiogenesis In Vitro

To begin, seed 100 microliters of HUVEC cells into the wells of a 96 well plate. Place the plate in a cell culture incubator at 37 degrees Celsius under 5%carbon dioxide for six hours to facilitate cell settling. Next, replace the medium in each well with 100 microliters of endothelial cell growth basal medium, containing 2%FBS, and incubate again.The following day, position the plate in a 96 well wound maker tool. Press down the lever of the device to generate a scratch. Carefully lift off the lid of the wound maker tool before releasing the lever to prevent double scratching.Wash the wells two times with 200 microliters of FBS-supplemented endothelial cell growth basal medium. After confirming total debris removal, pipette 100 microliters of the prepared stimulation or control solutions into each well. Use a live cell imaging microscope to acquire images every hour for 24 hours.The positive control showed successful migration in the original scratched area. Technical issues were seen as patchy cell growth, double scratching, or waves in the scratch borders. A 25%increase in relative wound distance was considered optimal.Incorrect separation of the positive and negative controls indicated low dynamic range.

Spheroid Sprouting Assay of Human Umbilical Vein Endothelial Cells (HUVECs) for Investigating Angiogenesis In Vitro 

To begin, add HUVECs into a 10 milliliter Falcon tube containing eight milliliters of endothelial growth medium. Add two milliliters of METHOCEL stock solution into the tube and shake well. Next, transfer 25 microliters of the mixture onto the inverted inner surface of a large, square Petri dish.Gently rotate the lid by 180 degrees and place it on the bottom of a cell culture vessel and incubate overnight. The next day, add 2.3 milliliters of rat tail collagen Type I to a vial containing 0.28 milliliters of 10X Medium 199. Keep this mixture on ice and mix until it is evenly yellow.Titrate the mixture against sodium hydroxide until it turns orange. Then, add 50 microliters of HEPES buffer to the final product. Collect the hanging cultured cells into a 50 milliliter Falcon tube with 20 milliliters of PBS, then centrifuge.After discarding the supernatant, add 0.1 milliliters of FBS and 0.4 milliliters of endothelial basal medium to the spheroid pellet. Gently tap the tube to resuspend the pellet. Now, pipette two milliliters of METHOCEL stock solution and mix well.Then, add two milliliters of the prepared collagen mixture to the resuspended spheroids. Dispense 0.5 milliliters of the resulting mixture into the wells of a 24-well plate and incubate. Next, pipette 100 microliters of diluted recombinant human vascular endothelial growth factor into the wells of the plate.Incubate the cell culture at 37 degrees Celsius under 5%carbon dioxide supplementation for 12 hours. The next day, with an inverted microscope, capture the images of the spheroids that are not in contact with the well rim, with each other, or show signs of damage. The spheroids stimulated with only basal medium or recombinant human vascular endothelial growth factor were clearly distinguishable.Spheroids in low-quality gels appeared to drop to the ground and dispersed. The negative control showed a moderate baseline sprouting rate while the human vascular endothelial growth factor doubled or tripled the relative sprouting length. The number of sprouts showed a similar dynamic range.

Immunocytochemistry of Human Umbilical Vein Endothelial Cell (HUVEC) Spheroids in a 3D Collagen Matrix for Investigating Angiogenesis In Vitro

To begin, fix the sprouted spheroid HUVEC gels in one milliliter of 4%paraformaldehyde for one hour. Wash the gels gently in one milliliter of PBS about three to four times. Detach the gels fully from the surface of each well of the plate, then transfer them into new 24 well plates.Add one milliliter of blocking solution to the well plate, then incubate on an orbital shaker for one hour at room temperature. After incubation, pipette 500 microliters of the primary antibody solution into the wells before incubation. The next day, wash the gels gently with PBS for five wash cycles of five minutes each.Add 500 microliters of the corresponding secondary antibody diluted with phalloidin-FITC in blocking buffer. Incubate the plate overnight at four degrees celsius on an orbital shaker. After washing the gels in PBS, transfer them onto microscope slides.Add two drops of DAPI-containing mounting medium onto a cover slip and place it over each gel. Seal the dried gels with nail polish. Place them in the dark before storing at four degrees Celsius until further analysis.