Name: an in vitro 3d model and computational pipeline to quantify the vasculogenic potential of ipsc-derived endothelial progenitors
Uploaded: 2019-05-13T13:00:00
Description: Watch this Scientific Journal Video about An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors at JoVE.com

This work was supported by the American Heart Association (grant number 15SDG25740035, awarded to J.Z.), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (grant number EB007507, awarded to C.C.), and the Alliance for Regenerative Rehabilitation Research &#38; Training (AR3T, grant number 1 P2C HD086843-01, awarded to J.Z.). We would like to acknowledge Prof. Jeanne Stachowiak (The University of Texas at Austin) for her technical advice on confocal microscopy. We are also grateful for discussions with Samuel Mihelic (The University of Texas at Austin), Dr. Alicia Allen (The University of Texas at Austin), Dr. Julie Rytlewski (Adaptive Biotech), and Dr. Leon Bellan (Vanderbilt University) for their insight on the computational analysis of 3D networks. Finally, we thank Dr. Xiaoping Bao (University of California, Berkeley) for his advice on differentiating iPSCs into iPSC-EPs.

1. Preparation of culture media and coating solutions
<ol>
	<li>To prepare vitronectin coating solution, dilute vitronectin 1:100 in Dulbecco&#8217;s phosphate-buffered saline (DPBS). 
	CAUTION: Once diluted, it is not recommended to store this solution for future use.</li>
	<li>Upon receiving phenol red-free, growth factor-reduced gelatinous protein mixture (see Table of Materials) from the manufacturer, thaw on ice at 4 &#176;C until the mixture is transparent and fluid. Keeping the mixture on i...

<ol>
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P., Micklem, K., Watt, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+for+quantifying+angiogenesis+in+the+Matrigel+assay+in+vitro.">A comparison of methods for quantifying angiogenesis in the Matrigel assay in vitro.</a> Tissue Engineering Part C: Methods. 17 (9), 895-906 (2011).</li><li>Rytlewski, J. A., Geuss, L. R., Anyaeji, C. I., Lewis, E. W., Suggs, L. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+endothelial+progenitor+differentiation+from+human+pluripotent+stem+cells+via+Wnt+activation+under+defined+conditions.">Directed endothelial progenitor differentiation from human pluripotent stem cells via Wnt activation under defined conditions.</a> Wnt Signaling: Methods and Protocols. 1481 (1), 183-196 (2016).</li><li>Crosby, C. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+vasculogenic+potential+of+iPSC-derived+endothelial+progenitors+in+collagen+hydrogels.">Quantifying the vasculogenic potential of iPSC-derived endothelial progenitors in collagen hydrogels.</a> Tissue Engineering Part A. , (2019).</li><li>Li, C. H., Tam, P. K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+iterative+algorithm+for+minimum+cross+entropy+thresholding.">An iterative algorithm for minimum cross entropy thresholding.</a> Pattern Recognition Letters. 19 (8), 771-776 (1998).</li><li>Kollmannsberger, P., Kerschnitzki, M., Repp, F., Wagermaier, W., Weinkamer, R., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+small+world+of+osteocytes:+Connectomics+of+the+lacuno-canalicular+network+in+bone.">The small world of osteocytes: Connectomics of the lacuno-canalicular network in bone.</a> New Journal of Physics. 19 (7), (2017).</li><li>Crosby, C. 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This protocol involves the long-term culture of cells in three types of cell culture media: E8, LaSR Basal, and EGM-2. Therefore, great care should be taken to appropriately sterilize all materials. Additionally, lab coats and ethanol-cleaned gloves should always be worn when working in the laboratory&#8217;s laminar flow hood. It is recommended to frequently test for mycoplasma contamination; if a large amount of debris is observed during iPSC culture or a sudden drop in differentiation efficiency is noted, mycoplasma c...

Human umbilical vein endothelial cells (HUVECs) and other primary endothelial cell types have been utilized for two decades to model blood vessel sprouting and development in vitro1. Such vascular platforms promise to illuminate molecular and tissue-level mechanisms of cardiovascular disease and may present physiological insight into the development of primitive vascular networks2,3. Though the field of vascular modeling has witnessed significant advances, a &#8220;gold standard&#8221; assay that can quantitatively model and assess physiological vascular development remains elusive. Most published protocols do not adequately recapitulate the vascular niche to encourage the formation of mature, functional blood vessels or do not have a method to quantitatively compare the vasculogenic potential of the assessed cell types in three dimensions (3D).
Many current vascular models are limited in their ability to mimic the physiological vascular niche. One of the most commonly employed in vitro platforms is the gelatinous protein mixture-based tube formation assay. Briefly, HUVECs are seeded as single cells on a thin layer of gel that consists of proteins harvested from murine sarcoma extracellular matrix (ECM); within one to two days, the HUVECs self-assemble into primitive tubes4. However, this process occurs in two-dimensions (2D) and the endothelial cells (ECs) utilized in this assay do not form enclosed, hollow lumens, thereby limiting the physiological significance of these studies. More recently, ECs and supporting cells (e.g., mesenchymal stem cells (MSCs) and pericytes) have been co-cultured in 3D microenvironments that simulate the fibrous architecture of the native ECM, such as collagen or fibrin hydrogels5. To model vascular development in this microenvironment, polymeric beads coated with ECs are typically employed6. The addition of exogenous growth factors and/or growth factors secreted by other cells interstitially embedded in the hydrogel can induce the ECs, coating the polymeric beads, to sprout and form single lumen; the number and diameter of sprouts and vessels can then be computed. However, these sprouts are singular and do not form an enclosed, connected network as is seen in physiological conditions and thus is more reminiscent of a tumor vasculature model. Microfluidic devices have also been utilized to mimic the vascular niche and to promote the formation of vasculature in EC-laden hydrogels7,8. Typically, an angiogenic growth factor-gradient is applied to the circulating cell culture medium to induce EC migration and sprouting. ECs that constitute the lumen of developed vessels are sensitive to the shear stress induced by the application of fluid flow through the microfluidic device; thus, these microfluidic devices capture key physiological parameters that are not accessible in the static models. However, these devices require costly microfabrication abilities.
Most importantly, all three vascular models (2D, 3D, microfluidic) overwhelmingly utilize primary ECs as well as primary supporting cell types. Primary cells cannot be developed into an effective cardiovascular therapy because the cells would engender an immune response upon implantation; furthermore, HUVECs and similar primary cell types are not patient-specific and do not capture vascular abnormalities that occur in patients with a genetic disposition or pre-existing health conditions, e.g., diabetes mellitus. Induced pluripotent stem cells (iPSCs) have emerged in the past decade as a patient-specific, proliferative cell source that can be differentiated into all somatic cells in the human body9. In particular, protocols have been published that outline the generation and isolation of iPSC-derived endothelial progenitors (iPSC-EPs)10,11; iPSC-EPs are bipotent and can, therefore, be further differentiated into endothelial cells and smooth muscle cells/pericytes, the building blocks of mature, functional vasculature. Only one study has convincingly detailed the development of a primary capillary plexus from iPSC-EPs in a 3D microenvironment12; though this study is critical to an understanding of iPSC-EP assembly and differentiation in natural and synthetic hydrogels, it did not quantitatively compare the network topologies of the resulting vasculature. Another recent study has used the polymeric bead model to compare the sprouting of HUVECs and iPSC-derived ECs5. Therefore, there is a clear need to further elucidate the physical and chemical signaling mechanisms that regulate iPSC-EP vasculogenesis in 3D microenvironments and to determine if these cells are suitable for ischemic therapy and cardiovascular disease modeling.
In the past decade, different open-source computational pipelines and skeletonization algorithms have been developed to quantify and compare vascular network length and connectivity. For example, Charwat et al. developed a Photoshop-based pipeline to extract a filtered, binarized image of vascular networks derived from a co-culture of adipose-derived stem cells and outgrowth ECs in a fibrin matrix13,14. Perhaps the most widely used topology comparison tool is AngioTool, a program published online by the National Cancer Institute15; despite the program&#8217;s widespread adoption and well-documented fidelity, the program is limited to analyzing vessel-like structures in two dimensions and other programs, including AngioSys and Wimasis, share the same dimensionality limitation16. Powerful software suites such as Imaris, Lucis, and Metamorph have been developed to analyze the network topology of engineered microvasculature; however, these suites are cost-prohibitive for most academic labs and limit access to the source code, which may hinder the ability of the end-user to customize the algorithm to their specific application. 3D Slicer, an open-source magnetic resonance imaging/computed tomography package, contains a Vascular Modeling Toolkit that can effectively analyze the topology of 3D vascular networks17; however, the analysis is dependent on the user manually placing the end-points of the network, which may become tedious when analyzing a large dataset and can be influenced by the user&#8217;s subconscious biases. In this manuscript, a computational pipeline that can quantify 3D vascular networks is described in detail. To overcome the above-outlined limitations, this open source computational pipeline utilizes ImageJ to pre-process acquired confocal images to load the 3D volume into a skeleton analyzer. The skeleton analyzer uses a parallel medial axis thinning algorithm, and was originally developed by Kerschnitzki et al. to analyze the length and connectivity of osteocyte networks18; this algorithm can be effectively applied to characterize the length and connectivity of engineered microvasculature.
Altogether, this protocol outlines the creation of microvascular networks in 3D microenvironments and provides an open-source and user-bias free computational pipeline to readily compare the vasculogenic potential of iPSC-EPs.

<table>
	<tbody>
		<tr>
			<td>&#181;-Slide Angiogenesis</td>
			<td>Ibidi</td>
			<td>N/A</td>
			<td>A flat, glass bottom tissue-culture plate with side walls enables facile confocal imaging</td>
		</tr>
		<tr>
			<td>96 well, round bottom, ultra low attachment microplate, sterile</td>
			<td>Corning</td>
			<td>7007</td>
			<td>Prevents the binding of cell-laden collagen hydrogels to the cell culture dish</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td>Gentle cell detachment solution; does not degrade extracellular epitopes vital for FACS</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo Scientific</td>
			<td>12634010</td>
			<td>The base media for iPSC-EP differentiation.&#160;</td>
		</tr>
		<tr>
			<td>Barnstead GenPure xCAD Plus&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>50136165</td>
			<td>Water purification system; others can be readily substituted</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution,7.5% in DPBS, sterile-filtered, BioXtra, suitable for cell culture</td>
			<td>Fisher Scientific</td>
			<td>A8412</td>
			<td>To preserve cell viability when FACs sorting</td>
		</tr>
		<tr>
			<td>CD34-PE, human (clone: AC136)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-140</td>
			<td>Antibody used for FACs isolation of iPSC-EPs</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>LC Laboratories</td>
			<td>C-6556</td>
			<td>Induces the formation of mesoderm from pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Collagen I Rat Tail High Protein 100 mg</td>
			<td>VWR</td>
			<td>354249</td>
			<td>Main component of the 3D microenvironment</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes (15/50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-959-49D/A</td>
			<td>Used to store and mix relatively large volumes of reagents and cell culture media</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>To counterstain and visualize cell nuclei</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320-082</td>
			<td>For dilution of Matrigel and thawing of pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>ThermoFisher</td>
			<td>14190-250</td>
			<td>To wash monolayer cultures</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E8008</td>
			<td>For passaging of pluripotent stem cell colonies and to prevent cell aggregation when FACs sorting</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2</td>
			<td>PromoCell</td>
			<td>C-22011</td>
			<td>Promotes endothelial cell viability and proliferation</td>
		</tr>
		<tr>
			<td>Essential 8 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517001&#160;&#160;</td>
			<td>For maintenance of pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Glycine,BioUltra, for molecular biology, &#62;=99.0% (NT)</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>Neutralizes remaining detergent</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate,&#62;=95%</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>Component of iPSC-EP differentiation medium</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>1.8.0_152</td>
			<td>Multi-paradigm numerical computing environment (free available at most academic institutions)</td>
		</tr>
		<tr>
			<td>Matrigel Matrix GFR PhenolRF Mouse 10 mL (gelatinous protein mixture)</td>
			<td>Fisher Scientific</td>
			<td>356231</td>
			<td>Diluted in DMEM/F12 to coat plates for iPSC-EP differentiation</td>
		</tr>
		<tr>
			<td>Medium-199 10X</td>
			<td>Thermo Fisher Scientific</td>
			<td>1825015</td>
			<td>Used to balance final hydrogel osmolarity and pH</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.7 mL)</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>Stores small volumes of reagents</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td>The main ingredient of the immunostaining solutions</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Antibiotic used after sorting to remove possible contamination from FACS instrument</td>
		</tr>
		<tr>
			<td>Recombinant Human VEGF 165 Protein</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td>Mitogen that stimulates endothelial cell proliferation and tubulogenesis</td>
		</tr>
		<tr>
			<td>Rhodamine phalloidin</td>
			<td>Themo Fisher Scientific</td>
			<td>R415</td>
			<td>To identify F-actin deposition and therfore outline the borders of the vascular networks</td>
		</tr>
		<tr>
			<td>Triton X-100 (nonionic surfactant)</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td>Detergent used to gently permeabilize cells</td>
		</tr>
		<tr>
			<td>Tween-20 (emulsifying reagent)</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td>Increases the binding specificity of the added antibodies</td>
		</tr>
		<tr>
			<td>VE-Cadherin (F-8)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9989&#160;</td>
			<td>To identify 3D endothelial lumen in collagen hydrogels</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>ThermoFisher</td>
			<td>A14700</td>
			<td>For maintenance of pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td>Preserves pluripotent stem cell and iPSC-EP viability when dissociated and re-seeded</td>
		</tr>
	</tbody>
</table>

an in vitro 3d model and computational pipeline to quantify the vasculogenic potential of ipsc-derived endothelial progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

After differentiation (Figure 1), FACS&#160;and encapsulation of iPSC-EPs in collagen hydrogels, the cells will typically remain rounded for 24 h before beginning to migrate and form initial lumens. After about 6 days of culture, a primitive capillary plexus will be visible in the hydrogel when viewed with brightfield microscopy (Figure 2). After imaging the fixed, stained cell-laden hydrogels on a confocal microscope (Mo...

Watch this Scientific Journal Video about An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors at JoVE.com

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Our protocol enables unique insights into the molecular and bulk tissue level mechanisms of vasculogenesis that may aid in the engineering functional vasculature for cardiovascular disease therapy in modeling. This protocol allows the creation of robust, three-dimensional vascular networks for evaluating the vasculogenic potential of various platforms, and also provides a free, open source computational pipeline for analyzing final network topology. Begin by adding 250 microlitres of cell detachment solution per well of the D5D6 differentiated cell culture for 10 minutes at 37 degrees Celsius.At the end of the incubation, use a P1000 pipette tip to dissociate the cells into single cell suspensions. Pool the cell solutions in a single 15 milliliter conical tube for centrifugation. We suspend the pellet in 200 microliters of ice cold sorting buffer and label the cells with five microliters of concentrated fluorescence conjugated CD34 antibody for 10 minutes at four degrees Celsius.At the end of the incubation, wash the cells in five mililitres of ice cold sorting buffer. Filter the suspension through a 40 micrometer pour strainer into a five milliliter fluorescence activated cell sorting, or FACS, tube. Before running the sample, sort one times 10th of the fourth unlabeled cells on the fluocitometer, gating a region at a high fluorescence intensity that does not contain any of the unlabeled cells to serve as the negative control.Then sort the labeled sample contain endothelial progenitors derived from induced pluripotent stem cells based on their high CD34 expression. At the end of the sort, transfer the endothelial progenitor cells to a microcentrifuge tube for centrifugation. For collagen hydrogel encapsulation of the progenitor cells, we suspend the sorted cell pellet in 200 microliters of Endothelial Growth Medium 2, supplemented with 10 micromiliter of the ROCK Inhibitor Y-27632.Add the cells to 400 microliters of seeding medium in a 1.8 mililiter microcentrifuge tube on ice. Mix 350 microliters of collagen into the cell suspension. The solution will become pale yellow.Next, mix ten microliters of 1-Muller sodium hydroxide into the collagen and cell solution. The solution will now become bright pink. Pipette 56 microliters of this neutralized collagen cell solution into individual wells of a 96 well ultra-low attachment U-bottom cell culture plate for a 30-minute incubation at 37 degrees Celsius.At the end of the incubation, examine the wells under a bright field microscope to check that the cells have been evenly distributed. Then add 100 microliters of freshly prepared Endothelial Growth Medium 2, supplemented with ROCK Inhibitor and Vascular Endothelial Growth Factor to each well and return the plate to the 37 degree Celsius incubator. After one week of culture, add 250 microliters of 4%paraformaldehyde to one well of a 48 well plate per hydrogel and remove the medium from the hydrogels.Then use fine-tipped tweezers to transfer the hydrogels to the paraformaldehyde containing wells. After 10 minutes at room temperature, rapidly wash the hydrogels with PBS and permeabilize the 3-D cultures with 250 microliters of a 0.5%non-ionic surfactant for five minutes at room temperature. Wash the hydrogels with two five minute washes at room temperature with 250 microliters of PBS, supplemented with 300 milliliter glycine per wash, followed by the immersion of each hydrogel in 250 microliters of blocking buffer for 30 minutes at room temperature.Label the cells with the appropriate primary antibodies diluted in blocking buffer overnight at four degrees Celsius, followed by two washes in 0.5%emulsifying reagent and Dulbecco's PBS. After the second wash, label the cells with the appropriate species specific secondary antibody for two hours at room temperature protected from light, before washing the 3-D cultures two times in 0.5%emulsifying reagent and Dulbecco's PBS, as demonstrated. To visualize the cell nuclei, add DAPI, diluted at a one to 10000 concentration in PBS to the samples for a two minute incubation at room temperature, followed by two washes in 0.5%emulsifying reagent and Dulbecco's PBS.Then use fine-tipped tweezers to transfer each sample to an appropriate viewing vessel. After differentiation, sorting, and encapsulation in collagen hydrogels, the cells will typically remain rounded for 24 hours, before beginning to migrate and form initial lumens. After about six days of culture, a primitive capillary plexus will be visible in the hydrogel when viewed with bright field microscopy.After imaging the fixed, stained, cell laden hydrogels on a confocal microscope, the pre-processed images are converted to a skeleton which enables an analysis of the overall length and connectivity of the network. It is imperative to add antibiotics after FACS, as sterilizing the interior of this instrument is difficult, and to remove the antibiotics 24 hours after cell encapsulation. Using this technique, we were able to determine which physical and chemical characteristics extracellular matrix mimicking biomaterials govern the vasculogenic potential of iPSC-derived derived endothelial progenitors.

Research

JoVE Journal

Developmental Biology

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Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

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