Name: Quantifying the Endothelial Cell Network Formation After the 3D Stromal-Endothelial Cell Co-Culture
Uploaded: 2023-05-19T14:50:00
Duration: 2 min 34 s
Description: Watch this Scientific Journal Video about Quantifying the Endothelial Cell Network Formation After the 3D Stromal-Endothelial Cell Co-Culture at JoVE.com

quantifying the endothelial cell network formation after the 3d stromal-endothelial cell co-culture

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

The bone and bone marrow are structurally and functionally complex organs central to human health. This is reflected by the existence of distinct niches that regulate hematopoiesis and bone maintenance1. It is now widely accepted that in healthy bone marrow, the maintenance and expansion of hematopoietic and skeletal stem cells, as well as their progeny, are controlled by distinct niches. These niches comprise various cell types, including osteolineage cells, mesenchymal stem cells, endothelial and perivascular cells, neuronal and glial cells, adipocytes, osteoclasts, macrophages, and neutrophils2. Not surprisingly, these mostly vasculature-associated niches are also involved in the development of various types of leukemia3 and are the site of metastasis for different cancers4. Owing to its specific roles in bone formation, remodeling, and bone (marrow) maintenance, the bone-associated vasculature has distinct specialized structures different from the vasculature found elsewhere in the body5,6,7. Thus, anti-angiogenic or vasculature-modulating drugs applied systemically may have different effects within these specialized environments8. Therefore, models to investigate the molecular mechanisms involved in maintaining the bone and bone marrow physiological properties, bone and bone marrow regeneration, and responses to therapeutic treatments are highly desirable.
Classical two-dimensional (2D) tissue cultures and in vivo investigations using animal models have provided invaluable insight into the roles of different cells and molecular players involved in the development of bone and bone marrow9,10. Models that allow for high-throughput experiments with relevant human cells could improve our understanding of how to modulate selected parameters in these highly complex systems.
In the past decade, principles derived from tissue engineering have been employed to generate 3D tissue models11,12. These have mostly relied on the encapsulation of tissue-relevant cells into biomaterials to establish 3D mono- or co-cultures13. Among the most used biomaterials are fibrin14, collagen15, and Matrigel16,17, all of which are highly biocompatible and provide appropriate conditions for the growth of many cell types. These biomaterials have the ability to generate in vitro models that recapitulate key aspects of the different vascular niches found in vivo18. Moreover, the use of microfluidic devices to generate perfused vascular bone and bone marrow models has contributed to the generation of in vitro models of higher complexity19,20,21,22.
The difficulty in controlling the composition and engineering the properties of naturally occurring biomaterials has inspired the development of synthetic analogs that can be rationally designed with predictable physical, chemical, and biological properties23,24. We have developed fully synthetic factor XIII (FXIII) cross-linked poly(ethylene glycol) (PEG)-based hydrogels, which are functionalized with RGD peptides and matrix metalloprotease (MMP) cleavage sites to facilitate cell attachment and remodeling25,26. The modular design of these biomaterials has been successfully used to optimize conditions for the formation of 3D vascularized bone and bone marrow models27,28.
For the testing of larger numbers of different culture conditions and new therapeutics, models with a higher throughput capability are required. We have recently shown that the FXIII cross-linking of our PEG hydrogel can be controlled through an electrochemical process such that an in-depth hydrogel stiffness gradient is formed29. When cells are added on top of such hydrogels, they migrate toward the interior and gradually develop into highly interconnected 3D cellular networks30. The elimination of the need to encapsulate cells into the hydrogel, which is usually present with other 3D scaffolds, not only simplifies the experimental design but also allows the sequential addition of different cell types at different time points to generate complex co-culture systems. These hydrogels are available pre-cast onto glass-bottom 96-well imaging plates, thus making the establishment of 3D cultures achievable by manual as well as automated cell seeding protocols. The optical transparency of the PEG hydrogels renders the platform compatible with microscopy.
Here, we present a straightforward method for the generation and characterization of vascularized osteogenic niches within this&#160;ready-to-use, synthetic plug-and-play platform. We show that the development of vascular networks can be stimulated with a growth factor commonly used for inducing in vitro osteogenesis, bone morphogenetic protein-2 (BMP-2), while osteogenic differentiation can be prevented by supplementation of fibroblast growth factor 2 (FGF-2)27,31. The networks formed are different when compared to FGF-2-stimulated networks in terms of the overall appearance, as well as the cell and ECM distribution. Moreover, we monitored the osteogenic induction using alkaline phosphatase as a marker. We demonstrate the increased expression of this marker over time and compare the expression to that in FGF-2 stimulated networks using qualitative and quantitative methods. Finally, we demonstrate the suitability of the generated niches of this model for two potential applications. Firstly, we performed a proof-of-concept drug sensitivity assay by adding bevacizumab to pre-formed niches and monitoring the degradation of the vascular networks in its presence. Secondly, we added MDA-MB-231 breast cancer and U2OS osteosarcoma cells to pre-formed osteogenic niches, showing that the niches can be used to study interactions between cancer cells and their environment.

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

Most in vitro vascularization studies use naturally-derived matrices. While typically very conductive to biological processes, their inherent biological activity complicates the study of influences of single parameters. Here, we can selectively add our players of interest and assess how the system changes.The sequential seeding of cells on our blank slate synthetic hydrogels enables the formation of very defined niches. Here, hBMMSs colonize the hydrogels and deposit their inherent extracellular matrix. This provides the substrate for the subsequently seeded endothelial cells.This feature is not commonly found in other 3D in vitro models. The possibility to add different cell types sequentially allows for the creation of high-throughput compatible in vitro models with great complexity. These models can be adapted to generate more specific osteogenic vascularized niches and to investigate the functions of cellular and molecular players.

Watch this Scientific Journal Video about Quantifying the Endothelial Cell Network Formation After the 3D Stromal-Endothelial Cell Co-Culture at JoVE.com

Quantifying the Endothelial Cell Network Formation After the 3D Stromal-Endothelial Cell Co-Culture  

After establishing the stromal endothelial cell co-culture, acquire the GFP signal from the GFP huvec using fluorescence microscopy with settings suitable for quantification. Pre-process all the images acquired on the same day to further enhance the contrast. If using fiji or image J open all the enhanced GFP channel images at the same time point and open the brightness and contrast menu.Select an image representing an intermediate condition and auto adjust the contrast by selecting auto. Click set and check propagate to all other open images. Visually assess whether the automatically selected range fits all the images of the current time point.If needed, manually readjust and repropagate the range to all the images and save the adjusted images as TIFF files. Next, apply a median blur filter to all the images. Reduce the size by binning and save them as gray scale RGB color TIFF files in a folder for quantification.This could be done manually or in batch mode using macros. Analyze all the created images using the batch process mode in the angiogenesis analyzer for image J.Then validate the quantification results by examining the overlays of the recognized structures and the original images. Adjust pre-processing parameters, reanalyze original images, or exclude problematic areas if the algorithm detects artificial structures with few or no cells visible in the original image.Finally normalize the obtained values to an area of one square millimeter by multiplying the values of each sample by the ratio of the analyzed area to one square millimeter. Quantified parameters of the GFP huvec networks showed that the total network length was highest in the presence of FGF-2 and lowest in the absence of growth factors. The number of junctions indicating branching points in the networks followed the same trend as the total length.Conversely, both growth factors featured significantly fewer isolated segments indicating higher interconnectivity than the condition without any growth factor.

quantifying-endothelial-cell-network-formation-after-3d-stromal

Research

JoVE Journal

Bioengineering

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

Establishing the 3D Stromal Monoculture

To begin, take a tube of hBM-MSCs and pellet them by centrifugation. Discard the supernatant and resuspend the pellet in an appropriate volume of basal medium. Then, prepare 50 milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors.Finally, add the hBM-MSCs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per milliliter. Remove the polypropylene adhesive film covering the 96 well hydrogel plate to seed the plate. Carefully aspirate the storage buffer covering the hydrogels by positioning the aspirator tip against the wall of the well and slowly moving toward the edge of the inner well.While seeding, mix the cell suspension periodically to keep the mixture homogenous and add 200 microliters of the cell suspension to each well of the plate. Then, maintain the cultures at 37 degrees Celsius and 5%carbon dioxide in a humidified atmosphere. Alternatively, use an automated plate washer with the aspiration nozzles set at least 3.8 millimeters from the plate carrier and toward the edge of the well and aspirate the storage buffer.Seed the plate by mixing the cells with a serological pipette and dispensing equal numbers of cells to the well of the plate. Monitor the development of the cultures by Bright-field microscopy with a five times objective and acquire reference images approximately 30 minutes after seeding to evaluate the number of cells added.

Establishing the 3D Stromal-Endothelial Cell Co-Culture

To begin, take the pellet of the GFP-HUVECs grown to a confluence level of 80 to 100%and resuspend the cells in an appropriate volume of basal medium to achieve a concentration of one times 10 to the seventh cells per milliliter of stock solution. Prepare 50-milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors. Add the GFP-HUVECs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per microliter.Then aspirate the medium from the plate containing the stromal monoculture and add 200 microliters per well of the prepared GFP-HUVECs suspension. Incubate at 37 degrees Celsius in 5%carbon dioxide in a humidified atmosphere, changing the medium every three to four days. Monitor the development of the culture by bright-field and fluorescence microscopy using a five times objective.Differences between the cultures could be observed from the bright-field and fluorescence images showing only the GFP-HUVECs. The cultures appeared less developed without any growth factor, However, faster development was observed in the presence of FGF-2. In contrast, the bright-field images showed the densest cultures in the presence of BMP-2.However, vascular like networks formed in both growth factor-containing conditions and the most extensive and interconnected networks were formed with FGF-2.

Assessing the Osteogenic Differentiation of 3D Stromal-Endothelial Cells by Monitoring the Alkaline Phosphatase Activity

To begin, take the hBM-MSCs and GFP-HUVECs co-culture and wash once with 200 microliters per well of PBS. Add the BCIP/NBT substrate solution and incubate the cultures at 37 degrees Celsius while periodically monitoring the color development. Once the color develops in non-osteogenic conditions, immediately washed with 200 microliters per well of PBS.After fixing the samples in 4%paraformaldehyde, acquire color images of the stained samples. To quantify the ALP activity in cell lysates, add 200 microliters per well of 0.25%Trypsin-EDTA to the cultures washed with PBS and incubate at 37 degrees Celsius. Agitate the cultures every 10 minutes by vigorously pipetting up and down to facilitate digestion and monitor the culture morphology with a standard cell culture microscope.Then transfer the samples to 2-milliliter tubes and add 200 microliters of mesenchymal stem cell basal medium to inhibit the Trypsin. After centrifusing and discarding the supernatant, freeze the pellets at 80 degrees Celsius or proceed directly to the next step. Thaw the cell pellets, add 500 microliters of lysis buffer, and incubate for 30 minutes on ice.Next centrifuge at 16, 100 G for 10 minutes at four degrees Celsius and keep the samples on ice. Without disturbing any pelleted debris, dispense 50 microliters of the supernatant into each well of a transparent tissue culture 96-well plate in duplicates. Add 50 microliters of the prepared alkaline phosphatase reagent to the wells of the 96-well tissue culture plate.Shake the plate briefly and incubate it at 37 degrees Celsius for 10 minutes protected from light. Stop the reaction by adding 100 microliters of 1-molar sodium hydroxide using a multi-channel pipette. Using a plate reader, read the optical density at 410 nanometers.For DNA quantification, thaw the frozen samples, centrifuge them, and place them on ice. Without disturbing any pelleted debris, dispense 50 microliters of the cell lysate supernatant into the wells of a black 96-well plate in duplicates. Add the DNA standards in duplicates.Next, use a multi-channel pipette to add 50 microliters of the DNA staining agent, then incubate and read the fluorescence intensity according to the manufacturer's instructions. Utilize the standard curve values to determine and apply the conversion of the measured intensity values to the DNA concentrations. Finally, normalize the alkaline phosphatase values by dividing them by the respective DNA concentration of each sample.Quantification of alkaline phosphatase activity to characterize the osteogenic potential of the cultures showed that the normalized alkaline phosphatase activity varied greatly across conditions with a trend of increasing activity with higher concentrations of BMP-2 and a plateau at 100 nanograms per milliliter. The lowest activity levels were identified for the condition containing 50 nanograms per milliliter FGF-2. More extensive and intense purple staining was observed in the presence of BMP-2 as compared to FGF-2.