Using ex ovo chick chorioallantoic membrane (CAM) assay to

Methods/Protocols Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3190−3200

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Using ex Ovo Chick Chorioallantoic Membrane (CAM) Assay To Evaluate the Biocompatibility and Angiogenic Response to Biomaterials Naşide Mangir,†,‡ Serkan Dikici,† Frederik Claeyssens,† and Sheila MacNeil*,† †

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Kroto Research Institute, Department of Material Science and Engineering, University of Sheffield, Broad Lane, S3 7HQ Sheffield, United Kingdom ‡ Royal Hallamshire Hospital, Department of Urology, Glossop Road, S10 2DL Sheffield, United Kingdom S Supporting Information *

ABSTRACT: Biomaterials need to be vigorously tested at every stage of preclinical development. As demand for in vivo culture environments continues to increase, traditional animal models are often technically complex, ethically undesirable, time-consuming, and resource intensive and thus present a barrier to high throughput screening. The chick chorioallantoic membrane (CAM) assay has long been used to study the effects of drugs on angiogenesis in vivo, providing researchers with a readily available, accessible, self-sustaining, and high throughput screen without requiring animal facilities. It has also been recognized as an in vivo assay to test initial tissue response to biomaterials; however it has not yet gained widespread acceptance. This could be due to lack of specific protocols on how to optimize this assay to specifically test biomaterials. Here we describe how the ex ovo (shell-less) CAM assay can be effectively used to study the angiogenic potential and initial tissue response to biomaterials. In comparison to alternative in vivo approaches, this technique provides additional advantages to the researcher as it allows better visualization of implanted biomaterials and the ability to implant several samples simultaneously enabling combinatorial biomaterial assays to be conducted. KEYWORDS: chick chorioallantoic membrane (CAM) assay, biocompatibility, angiogenesis, biomaterials

1. INTRODUCTION

The CAM assay can be a valuable assay to test biomaterials extensively in vivo before they are further investigated in relevant animal models.1 In the context of biomaterials testing, the CAM can be effectively used as a short-term host for grafted materials, organs, and tissue samples where the angiogenic response and their safety and biocompatibility can be studied.2 It is also promising that the CAM assay has recently been demonstrated to produce data that is comparable to mouse assays in testing biodistribution and in vivo stability of radiopharmaceuticals.3 For the purpose of biomaterials testing, in ovo4 and ex ovo5,6 culture methods have been used. The ex ovo (embryo cultured outside of the egg shell) modification of the classical in ovo CAM assay (embryo cultured inside of the egg shell) offers several unique advantages for biomaterials testing. The ability to grow ex ovo cultures with comparable survival rates was first reported in 1974.7 Although it was reported by several authors that the ex ovo modification is associated with worse survival rates,8 our experience does not confirm this finding. We consistently have

Biomaterials need to be tested vigorously using in vivo assays at all stages of preclinical development. Because biomaterials comprise a wide range of natural and synthetic materials that are often combined with a range of bioactive molecules, proteins, or cells, the biocompatibility testing has to examine each of these constituents separately and in various combinations. This creates a demand for reproducible and technically simple bioassays that allow higher throughput screening of biomaterials prior to animal testing. The chorioallantoic membrane (CAM) of the chick embryo is an extraembryonic membrane that functions as an organ for gas exchange between the embryo and the environment. It is home to many blood vessels with a dense capillary network, and because it stays on top of the developing embryo, it is easily accessible for experimental interventions. The CAM assay has traditionally been used to test pro- and antiangiogenic response to drugs and to study many aspects of tumor angiogenesis.1 With recent advances in biomaterials science and engineering, another area where the CAM assay can prove useful is in biomaterials testing. © 2019 American Chemical Society

Received: February 3, 2019 Accepted: May 30, 2019 Published: May 30, 2019 3190

DOI: 10.1021/acsbiomaterials.9b00172 ACS Biomater. Sci. Eng. 2019, 5, 3190−3200

ACS Biomaterials Science & Engineering

Methods/Protocols

• Check for any cracked or damaged eggs and dispose of them in line with local institutional guidance. • Store the fertilized eggs at 10 °C for 10−12 days if not planning to start the incubation immediately. Tip: The fertilized eggs can be stored at 10 °C for up to 10−12 days before starting the incubation. Tip: The experiments can be planned beforehand so that the day of cracking the eggs (day 3), the day of implantation of test samples (day 7), and the day of sacrificing the embryo cultures (day 14) are all on weekdays. For example, if the embryo cultures are started on a Friday (day 0), the eggs are cracked on Monday (day 3), the test samples are implanted on Friday afternoon (day 7), and the embryos are sacrificed on the third Friday (day 14). This plan also allows the opportunity to inspect implanted samples between days 10 and 14 if needed. 3.2. Preparation of Fertilized Eggs for Incubation (days 0−3). • Do the following steps in a laminar flow cabinet. • Clean the rotating incubator parts by spraying IMS (70%) and leave all the parts to dry for 1−2 h in the laminar flow cabinet. • Gently clean the dirt, feathers, and excrement from the egg shells with IMS (20%) sprayed tissue paper. • Place the fertilized eggs in the rotating egg incubator horizontally. • Place a cup of sterile water in the incubator if there is no other means to provide humidity. • Incubate the eggs at 37−38 °C and 40−60% humidity while rotating 12 times a day. • Incubate the eggs for 3 days. Tip: The number of eggs that can be incubated depends on the number of experimental groups in hand, the size of the incubator, and the level of experience with the technique. It needs to be taken into account that not all of the ex ovo cultures will survive to the end of the experiments. Tip: In the authors’ experience using 3 dozen eggs for each experiment works well allowing 4−5 experimental groups with n = 6 embryos per group with an 80−85% survival rate. 3.3. Cracking the Egg Shells and Starting the ex Ovo Cultures (Day 3). • Do these steps under laminar flow cabinet. • Disinfect the weighing boats by dipping them in a bath of 70% IMS and leave them to dry. • Put 2 mL of PBS+Pen/Strep into each weighing boat. • Bring the rotating incubator into a stationary state and wait for at least 10 min. • Mark the upper surface of the horizontally lying eggshells with a marker pen. The embryo lies at the top surface of the horizontally lying egg. This will ensure cracking the egg at the exact opposite side, as far away from the embryo as possible. • Remove the eggs from the rotating incubator and place them horizontally on a stand with the marked line always facing upward. • Wear nonsterile gloves and wipe with alcohol, let them dry. • Crack each egg by hitting it on a hard surface (e.g., a 1000 mL beaker) from below holding it horizontally with the pen mark on top. • Transfer the chick embryo by gently separating the two halves of the egg shell to the sides and upward. Keep the egg close to the bottom of the weighing boat to maintain a

survival rates above 80%.5,9,10 The main advantage of the ex ovo culture method is better visualization of the growing embryo and access to a larger area of the CAM to study angiogenesis. Additionally, the vascularization process could be observed at all times during the experiment. In this work, we describe how the ex ovo CAM assay can be used as a valuable asset in a biomaterials laboratory to test the angiogenic potential of biomaterials and the initial in vivo response to them.

2. MATERIALS AND REAGENTS 2.1. Equipment in the Egg Lab. • Rotating egg incubator (90° rotation per hour) (RCOM King SURO, P&T Poultry, Powys, Wales) • Stationary egg incubator 37.5 °C with 60−80% humidity • Laminar flow cabinet • A hot plate set to 40−42 °C • A fridge with a temperature set between 4 and 10 °C. • Image acquisition unit (a USB microscope) • Record book (or excel sheet) 2.2. Growing ex Ovo Cultures. 2.2.1. Reagents. • Fertilized eggs • Penicillin−streptomycin in PBS (1%) • Alcohol, 20% to disinfect the egg shells and 70% to disinfect the equipment 2.2.2. Apparatus. • 89 × 89 × 25 mm3 (100 mL capacity) square plastic weighing boats • 1000 mL beaker 2.3. Collecting Data. 2.3.1. Reagents. • An oil in water emulsion (we used commercially available hand lotions, such as Simple hydrating hand cream, Unilever, U.K.) • Formaldehyde 3.7% in PBS 2.3.2. Apparatus. • Tissue forceps and scissors • A digital camera • A transillumination box containing a light source • External light sources (side lamps) as required 2.4. For Microinjection. 2.4.1. Reagents. Rhodamine labeled Lens culinaris agglutinin (LCA) (Vector laboratories) 5 μg/mL PBS 2.4.2. Apparatus. • Hypodermic needle 30G • A 1 mL syringe • Cotton buds • A dissection microscope (may be required for less-experienced users while doing microinjection) • A fluorescence or confocal microscope or both

3. PROCEDURE 3.1. Receiving the Fertilized Eggs and Planning the Experiments. • Receive the eggs laid on the same day of dispatch (day 0) • Record the date of dispatch and date received in line with local institutional regulations. (Researchers are advised to follow their own local institutional policies on ethical use of animals in research. In the authors’ institution, the procedures are required to be registered with ethical review committee who authorize the premises and carry out regular inspections. Each user needs to be authorized to carry out these experiments, and the embryo cultures can only be maintained for up to 14 days.) 3191



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Figure 1. Graphical demonstration of egg cracking technique. (1) The egg is kept in a stationary position and the top surface is marked with a pen. The marked surface stays at the top at all times. (2) The egg shell is cracked at the bottom by hitting it onto a hard surface. The cracked egg is immediately brought into the weighing boat. (3) The egg shell is separated into two halves by pulling it sideways and upward using the thumbs. The egg shell is kept very close to the bottom of the weighing boat during this maneuver so that the egg white forms a cushion around the egg yolk. (4) All the egg content is smoothly transferred into the weighing boat.

Figure 2. Observation of a successful embryo transfer with an intact egg yolk and live embryo on embryonic development day 3 (left-hand image). An unsuccessful embryo transfer with a live embryo (middle image) and a successful embryo transfer with an intact egg yolk but a dead embryo (righthand image) can also be observed. Error bars represent 1 cm.

• •

• •

vacuuming effect between the egg contents and the weighing boat provided by the egg white (Figure 1). A successful embryo transfer into the weighing boat is confirmed by observing the intact egg yolk and the beating heart of the chick embryo (Figure 2). Put a sterile Petri dish lid to cover the weighing boat and place the ex ovo culture on the hot plate set at 37−38 °C to prevent the embryonic heat loss until they are put in the incubator. Keep the ex ovo cultures in a 37−38 °C stationary sterile incubator with 60% humidity for the rest of the experiment until day 14. Check daily whether • The chick embryos are alive or dead and keep a record of it. Discard any dead embryos immediately according to laboratory guidelines (for animal waste) to avoid infections in the incubator. • The chick embryos are developing normally (normal development defined as a chronological

age [days of incubation] = morphological age [Hamburger−Hamilton stage]) (Figure 3). The chronological age and morphological age sometimes are not in concordance with each other, which could be due to infection, poor temperature control inside the incubator, and the toxicity of the implanted material. Some of the abnormalities observed by the authors are depicted in Figure 4. Tip: Doing the above step will allow the researcher to calculate his/her survival rates, to improve his/her technique, and to control for any other factors that might have an effect on the experimental results. Additionally, the survival rates are required to be reported as per ARRIVE guidelines on “animal research: reporting in vivo experiments”.11 Tip: The best time to do the embryo transfer is when the embryo is mature enough to survive outside of the eggshell but small enough to allow embryo transfer without any damage to it. This is between EDD 3 and 4. Beyond EDD 4 it will be very 3192



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Figure 3. Demonstration of a mismatch between the chronological age and the morphological age of the developing embryos. On the left is an embryo at embryonic development day (EDD) 4, which is slightly underdeveloped with a morphological age of Hamburger and Hamilton (HH) 14−16. On the right an embryo of the same chronological age, EDD 4, with appropriate morphological age demonstrating limb bud development (arrow) and eye development (dashed arrow). Scale bars represent 5 mm in both images.

Figure 5. Graph showing how chick survival rates increase with experience. Survival of the chick embryos of one of the researchers in our group at the beginning of their training (first 2 batches), an intermediate stage (between 2 and 4 batches) and after acquiring experience (after 6 batches). The improvement in survival rates can be observed for the beginner, intermediate and experienced stages as 25%, 68%, and 83%, respectively. Please note that the first 3 days of embryonic life is in ovo (the embryo developing inside the egg) and the survival rates are calculated for only the eggs that were alive at the day of egg cracking (day 3) in these experiments.

difficult to do a successful embryo transfer without damaging the CAM. Tip: The authors crack no more than 4−10 eggs at a time to decrease the time when embryos are outside the incubator as they are very sensitive to cold. Critical point: Please be aware that there is a learning curve for growing ex ova cultures. See Figure 5 for our own data. Critical point: To locate the developing embryo inside the egg at EDD 3 before cracking it, alternative methods can be used such as using transilluminating light as described before.2,12 3.4. Implantation of Test Materials (Day 7−8). • Take the ex ovo cultures from the static incubator and place in a laminar flow cabinet. • Use the hot plate at 37 °C to minimize the time embryos are cold. • Use sterile forceps or tweezers to handle the biomaterials. • Place the test sample halfway between the embryo and the outer border of the CAM and between two large vessels (Figure 6). Critical point: It is important to be able to delineate the exact borders of the CAM and not implant the sample outside of the CAM (Figure 7).

Tip: The actual experimental intervention on the CAM is done at the earliest opportunity after the CAM becomes mature enough. This generally corresponds to the end of EDD 7 or early on EDD 8. The surface area of the CAM expands rapidly between EDD 4−5 and 10 (Figure 8). The capillary proliferation significantly declines after EDD 10, after which point the vascular network undergoes maturation.6 Critical point: Prepare the biomaterials being implanted on the CAM beforehand. The materials need to be sterile and ideally the same shape, size, and weight. This is important because the results will mainly be analyzed by image analysis. The authors of this work use circular shaped biomaterials with a diameter between 7 and 10 mm and with a maximum weight of 0.5 mg. Heavy or large biomaterials would be expected to be more likely to disrupt local blood circulation and can potentially affect survival rates. Critical point: After implanting biomaterials onto the CAM, it is very important to keep checking and recording the survival of the embryos and the response to the grafted biomaterial, especially if it contains cellular components. For example, when a tissue engineered skin substitute is implanted onto the CAM, some of these biomaterials can fail to graft on to the CAM tissue.

Figure 4. Examples of abnormal embryonic development. A twin egg with a live, larger embryo and a dead smaller embryo is seen on the left, and an embryo with three eyes is seen on the right. 3193



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Figure 6. Correct placement of the test sample on the CAM. The dashed lines show the borders of the CAM and colored circles show the possible locations for implantation of the biomaterial.

Figure 7. An example of (1) incorrect and (2) correct placement of the test sample on the CAM on embryonic development day (EDD) 7. Sample 1 is implanted outside of the CAM whereas sample 2 is placed correctly on the CAM. Arrows show the borders of the CAM. Incorrect placement of the test sample blocks normal expansion of the CAM, which can be seen to accumulate behind the sample at EDD 9. Test sample 2 moves together with expanding CAM. Scale bars 10 mm for all images.

This is an important finding for the researcher as it can give valuable information about the biocompatibility of the implanted biomaterial (Figure 9). Additionally, any infections with the test samples need to be noticed (Figure 10). 3.5. Evaluation of Angiogenic Responses to Biomaterials (Day 11−14).

• Make sure all the embryos are alive before taking images of the CAM−biomaterial complex as dead embryos will result in false negative results (Figure 11). • If possible, use a digital camera that is fixed at a constant height for all samples. 3194



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Figure 8. Development of the CAM after first appearing on embryonic development day (EDD) 5 (black arrows) and growing up to cover the whole surface of the square weighing boat on EDD 9 (Black and red scale bars represent 1 cm and 5 mm, respectively).

Figure 9. Demonstration of CAM assay being used as a host for biomaterials testing. In the upper raw images, the gross appearance and histologic sectioning of a successfully engrafted human tissue engineered (TE) skin equivalent is shown. A live, healthy, moist graft can be observed together with good tissue integration in the histology. In the lower raw images, a nonengrafted TE skin equivalent is shown as a discoloured, dehydrated, and thinned graft. Histologic evaluation of the nonengrafted sample shows a totally separated CAM and biomaterial with no tissue integration and apparently more fibrotic graft.

Critical point: Acquiring high quality images may initially be difficult and may require some trial and error. For imaging, we use a fixed camera on a transillumination box with two side lamps. The variability in images caused by different lighting is shown in Figure 12.

Tip: To improve the quality of the image a colored liquid (a 20% oil in water emulsion or methylene blue solution) can be injected just beneath the test sample and the CAM to conceal the unnecessary background (e.g., the body of the embryo) and 3195



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Figure 10. Appearance of an infected hydrogel implanted on the CAM. The hyphae (arrow) most probably indicate a fungal infection.

Figure 11. Demonstration of how overlooking dead embryos can result in false negative or false positive results. The left image shows a dead embryo with pale, dry, and poor vasculature all around the CAM. In the middle image, another dead embryo is shown; however this time the embryo has just died making it more difficult to understand. In the right image, a normal CAM looking bright, glossy, and moist with a normal distribution of blood vessels. (Scale bar represents 5 mm for all images)

Figure 12. Demonstration of how lighting problems can lead to variability in image quality. (A) Inadequate lighting throughout the image preventing proper observation of blood vessels growing toward the test sample. (B) Lighting of the area with use of transillumination from below results in even lighting; however because the sample lies between egg yolk and egg white, there is too much light on the right lower corner where the egg yolk ends, and the egg white causes strong transillumination. (C) A homogeneously illuminated sample with inadequate lightening. (D) a good quality image with good lighting from below and with two side lamps allowing good visualization and quantification of blood vessels. Scale bar represents 2 mm for all images.

3.6. Sacrificing the Embryo Cultures and the Retrieval of Samples (Day 14).

increase the contrast (Figure 13). To do this, a 20 mL syringe with a 21 gauge needle is used. Tip: Angiogenic responses to biomaterials are generally evaluated at EDD 14. An exception to this can be when testing and looking for an anti-angiogenic response. In this case, we found that the best time to evaluate an anti-angiogenic response (for example when testing hydrocortisone releasing poly(lactic acid) scaffold) would be between EDD 11 and 12.

• Sacrifice the embryos by either bleeding or decapitation immediately after acquiring the images. • Excise the CAM−biomaterial complex leaving a margin of 1 cm CAM around the biomaterial using tissue scissors and forceps. • Fix the samples in formaldehyde (3.7%). 3196



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Figure 13. Another way of facilitating the image analysis is injecting a contrast agent underneath the sample before imaging. In the upper raw images, injection of a white contrast agent (oil in water emulsion; in this case, Simple hydrating hand cream, Unilever, U.K.) is shown to effectively cover the interfering background of chick feather and even the smaller blood vessels become obvious for quantification. In the lower rawimages, the results of injection of a blue contrast (10% Trypan blue solution in PBS) is shown to effectively cover the unnecessary background caused by the embryo; however, the smaller blood vessels were not easily seen with a blue background in this case. Scale bars represent 5 mm for all images.

Critical step: It is important to note that the CAM of the chick embryo does not normally contain nerve endings; however, the embryo itself starts developing nerve endings after EDD 11. Therefore, handling the CAM alone will not give pain to the embryo, but any interventions to the embryo will require some form of analgesia/sedation particularly after EDD 11. Researchers are advised to follow their own institutional guidance on this. The Home Office guidance in the U.K. states that a prior sedation or anesthesia should be carried out before humane killing unless to do so would be likely to cause greater distress than using the same method of killing without sedative or anesthetic. One method could be exposing the embryo to cool temperatures.

the annulus are counted as 1 vessel, whereas those branching outside the annulus are considered to be 2 vessels.14 • Additionally, a semiautomated quantification method based on ImageJ software can be used.15 To do this, a free online plug in, NeuronJ (Meijering et al., Cytometry Part A 2004, 58A, 167−176; http://www.imagescience.org/ meijering/software/neuronj/), can be used. The images are first converted to grayscale (8-bit) and sharpened twice. Then all discernible vessels in the image are traced with the Neuron J tracing tool. This allows calculation of total vessel length, the total number of blood vessels, and small and larger blood vessels. This is described in detail previously.15 • A more detailed image processing can also be done via a combination of ImageJ and Adobe Photoshop software.16 Briefly, crop the area of interest and split the red, green, and blue channels using Adobe Photoshop (ADOBE Systems Inc., San Jose, California, USA). Then import only the green channel to ImageJ (Wayne Rasband, National Institutes of Health, USA) for unsharp mask filtering, enhancing the local contrast, noise removal, and segmentation. Finally, quantify the number of branch points using AngioTool software (National Cancer Institute, USA. To download: https://ccrod.cancer.gov/confluence/display/

4. QUANTIFICATION OF RESULTS 4.1. Macroscopic Evaluation of Angiogenic Responses. • The quantification of the angiogenic response is performed by first acquiring digital images. • Several methods have been described to quantify the angiogenic response using these digital images. • The authors frequently use the “vasculogenic index”.5,13 To do this all discernible vessels (capillaries, arterioles, venules) traversing a 1 mm annulus around a 2 mm imaginary circle drawn around the scaffold material are counted provided that they form an angle of less than 45° with a line radiating from the center. Vessels branching within 3197



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Figure 14. Flowchart detailing the image analysis technique that can be used to quantify results of the angiogenesis experiments. The final image that results from each step is demonstrated on the right-hand side.

Figure 15. Demonstration of how the angiogenic response to a biomaterial (ascorbic acid releasing electrospun poly(lactic acid) scaffold in this case) can be quantified on hematoxylin and eosin (H&E) stained frozen sections. The blood vessels underneath and adjacent to the scaffold (arrows) can be observed to have increased in A compared to B where a control scaffold was used. Scale bars represent 100 μm.

The total number of blood vessels on CAM adjacent to the biomaterials can be counted on standard hematoxylin and eosin (H&E) sections. Two independent researchers using two independent microscopes can count the vessels at 10× magnification. This is particularly useful for bigger blood vessels (Figure 15). 4.3. Evaluation of the Initial Tissue Response to Biomaterials. The CAM assay is less frequently used to evaluate tissue responses to biomaterials. This is because the immune system of the chick embryo only starts developing after

ROB2/Downloads). Average blood vessel lengths can also be calculated by using binary image histograms with known pixel/mm ratios in ImageJ software. Please see Figure 14. Tip: The key to getting accurate quantification is getting good quality images consistently. 4.2. Histologic Evaluation of Angiogenic Responses. Blood vessels can be counted on histologic sections of the CAM−biomaterial complex. 3198



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Figure 16. Characterizing the initial tissue response to different types of biomaterials. A mild inflammatory cell infiltration in the CAM area underneath the implanted poly(lactic acid) (PLA) scaffold can be observed in the upper row. An expanded view of this sample shows small blood vessels and cell infiltration from the CAM between the PLA fibers. In contrast to this, a gelatin based hydrogel triggers almost no tissue reaction on the CAM underneath the hydrogel with no cellular infiltration and no integration of hydrogel into the CAM, which is seen to easily detach from the CAM surface. An expanded view of this sample confirms almost no cellular infiltration by cells from the CAM and detachment of the hydrogel from the CAM without any damage. Scale bars represent 100 μm.

EDD 10. The first mononuclear phagocytes are detected in the chick embryo at EDD 9,17 and functional macrophages, which are responsible for the initiation of acute inflammatory response, are detectable in the circulation at EDD 14, the number of which suddenly increases at EDD 19.18 The lymphoid cells that constitute the cellular (T cells) and humoral immunity (B cells) were detected after EDD 11 and EDD 12, respectively.17 The chick embryo is considered immunocompetent only after EDD 18. Nevertheless, the CAM assay can give valuable information on the initial tissue response to biomaterials when the embryo cultures are run until EDD 14, as in our center; the value of the assay is limited to determining the early immune response only to implanted materials. The immune response to implanted materials and tissues has been described in detail elsewhere.1,2,19 In this methodology paper, we only demonstrate how we apply this knowledge to biomaterials testing (Figure 16).

an idea of the initial tissue response to the biomaterials. Thus, tissue engineered constructs, such as tissue engineered skin, can also be implanted and cultured on a readily available in vivo environment. However, it should be noted that researchers cannot rely on this model to characterize the immune response to the implanted material; this will require further animal studies. Other in vivo assays have also been used to study angiogenesis. Among these the mouse dorsal skin chamber (DSC) assay allows direct visualization of blood vessels at all times during the experiment with the use of intravital microscopy.20 This model can also accept xenogeneic grafts if nude mice are used, and it can provide information on the dynamics of blood vessel development. However, this Home Office regulated model is limited by the requirement for significant expertise and sophisticated equipment that may not be available in most laboratories. Furthermore, for biomaterials testing, implantation of the biomaterial will be difficult as the chamber lies vertically at the back of the mouse. Therefore, the ex ovo CAM assay stands out as an inexpensive, reproducible, and technically less challenging bioassay to allow high throughput screening.

5. SUMMARY The ex ovo CAM assay can be used as a readily available, reasonably high throughput assay to study the angiogenic potential of and initial tissue responses to biomaterials. The ex ovo (shell-less) modification gives very good survival rates, and with this technique a larger surface area of the CAM is available for all experimental interventions. The main advantage of this is that it allows implantation of several biomaterials on the same embryo at a time. Another advantage of the CAM is its ability to accept grafts that contain xenogeneic cells or proteins in the initial stages of embryonic life.10 This is because the immune system of the chick embryo starts developing only after day 11 of embryonic life, so when the embryos are cultured up to 14 days, this assay can give

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00172. Video of the procedure (MP4)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: s.macneil@sheffield.ac.uk. 3199



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ORCID

Adipose Derived Stem Cells to Promote Vascularization for Skin Wounds and Tissue Engineering. Biomaterials 2017, 129, 188. (16) Dikici, S.; Mangır, N.; Claeyssens, F.; Yar, M.; MacNeil, S. Exploration of 2-Deoxy-D-Ribose and 17β-Estradiol as Alternatives to Exogenous VEGF to Promote Angiogenesis in Tissue-Engineered Constructs. Regener. Med. 2019, 14 (3), 179−197. (17) Marga Janse, E. M.; Jeurissen, S. H. Ontogeny and Function of Two Non-Lymphoid Cell Populations in the Chicken Embryo. Immunobiology 1991, 182 (5), 472−481. (18) Friend, J. V.; Crevel, R. W. R.; Williams, T. C.; Parish, W. E. Immaturity of the Inflammatory Response of the Chick Chorioallantoic Membrane. Toxicol. In Vitro 1990, 4 (4−5), 324−326. (19) Ribatti, D. The Chick Embryo Chorioallantoic Membrane (CAM). A Multifaceted Experimental Model. Mech. Dev. 2016, 141, 70−77. (20) Michael, S.; Sorg, H.; Peck, C.-T.; Reimers, K.; Vogt, P. M. The Mouse Dorsal Skin Fold Chamber as a Means for the Analysis of Tissue Engineered Skin. Burns 2013, 39 (1), 82−88.

Naşide Mangir: 0000-0002-3062-6480 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge The Rosetrees Trust, U.K., and The Urology Foundation, U.K., for funding N.M. and the Republic of Turkey, The Ministry of National Education, for funding S.D. We also thank Prof. Richard Oreffo of University of Southampton for his generosity in giving us the initial training for the CAM assay.

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REFERENCES

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Using ex ovo chick chorioallantoic membrane (CAM) assay to

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