Alginate Encapsulation is a Highly Reproducible Method for Tumor Cell Implantation in Dorsal Skinfold Chambers
Year: 2005
Type: article
Abstract: BioTechniquesVol. 39, No. 6 BenchmarksOpen AccessAlginate encapsulation is a highly reproducible method for tumor cell implantation in dorsal skinfold chambersYong Wang, Qing Chen & Fan YuanYong WangDuke University, Durham, NC, USA, Qing ChenDuke University, Durham, NC, USA & Fan Yuan*Address correspondence to: Fan Yuan, Department of Biomedical Engineering, 136 Hudson Hall, Box 90281, Duke University, Durham, NC 27708, USA. e-mail: E-mail Address: [email protected] University, Durham, NC, USAPublished Online:30 May 2018https://doi.org/10.2144/000112082AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Transparent window chamber models have been used widely in chronic studies of microcirculatory phenomena, such as angiogenesis, microvessel remodeling, blood flow, and molecular transport (1–3). The most popular site for chamber implantation is the dorsal skin of mice or rats. To study tumor microcirculation, one needs to implant either a fragment of tumor tissues, a group of tumor spheroids, or a pellet of tumor cells into the center of the chamber to form a solid tumor. The choice of tissue fragments versus cultured spheroids or cells depends on the experience of the investigator as well as experimental design. In general, the advantage of using tissue fragments is that it is easy to control the distribution of tumor cells in the chamber. However, this procedure has two disadvantages. First, it is relatively expensive to maintain the tumor source in animals. Second, it is hard to control the number and the type of viable tumor cells inoculated into the chamber, because they depend on the tumor source and where the tissue fragment is taken from the source. Each tissue fragment is unique, since it is well known that tumor tissues are heterogeneous. The heterogeneity may cause significant variations in experimental observations. Alternatively, researchers have used tumor spheroids, which are less heterogeneous than tissue fragments, to produce a solid tumors in the chamber (4). However, the preparation of tumor spheroids is time-consuming, and it is difficult for some tumor cell lines to form spheroids (5,6). The other choice to produce tumors in transparent window chambers is to directly implant a dense pellet of tumor cells (7–9). Although it is easy to prepare the cell pellet, one potential problem in cell implantation is that tumor cells often spread randomly in the chamber within a short period after the implantation, presumably due to animal movement and inadequate adhesion between tumor cells. As a result, they cannot form a solid mass that grows gradually in the chamber. The cell spreading makes it difficult to monitor tumor growth and distinguish between newly formed microvessels versus host microvessels. It also reduces reproducibility and reliability of microcirculation studies. To this end, we proposed to encapsulate tumor cells in a polymer film and implant the film into the dorsal skinfold chamber.Two different polymers have been used in subcutaneous implantation of tumor cells: alginate (10) and Matrigel (11). Matrigel is a reconstituted basement membrane matrix that contains various growth factors such as epidermal growth factor (EGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF). These growth factors can induce angiogenesis in vivo (11), which may not be desirable in tumor angiogenesis studies. Alginate is a biocompatible and biodegradable natural polysaccharide and has minimal angiogenic effects (10). In addition, cell encapsulation in alginate gels is a well-developed technique in tissue engineering and bioprocessing (10,12,13). Finally, alginate has been shown in controlled drug release studies to have a pore size large enough to allow release of >50% of encapsulated proteins within 10 min (14), suggesting that alginate encapsulation is unlikely to prevent interactions between tumor and host cells via growth factors and cytokines. Therefore, alginate is a better choice for studying tumor cell-induced angiogenesis in the chamber.To test the feasibility of gel implantation in the dorsal skinfold chamber assay of tumor angiogenesis and microcirculation, we encapsulated 4T1 mouse mammary carcinoma cells in an alginate film and implanted the film in the dorsal skinfold chambers. The details of the chamber preparation have been well described in the literature (7,9). In brief, female Balb/c mice of approximately 20 g in body weight (Charles River Laboratory, Raleigh, NC, USA) were anesthetized with an intraperitoneal (i.p.) injection of a cocktail of ketamine and xylazine (80 mg ketamine and 10 mg xylazine per kilogram body weight). Then, titanium window chambers were surgically implanted on the dorsal skin flap of mice (7,8,15). After surgery, animals were allowed to recover for 48 h, since the chamber implantation caused disturbance of microvessels and microcirculation in subcutaneous tissues. The disturbance might interfere with tumor angiogenesis from host microvessels (see References 7 and 16). At the end of recovery, a dense cell pellet or the alginate film containing 4T1 cells was implanted in the center of the chamber. All procedures were performed under aseptic conditions. After surgery, the animals were kept ad libitum in a warm room with food and water and a 12-h light/dark cycle. The use of animals and the surgical procedures for chamber and tumor implantations were approved by the Duke University Institutional Animal Care and Use Committee.The alginate film containing 4T1 cells was prepared as follows. First, sodium alginate (Sigma, St. Louis. MO, USA) was dissolved in a sterile phosphate-buffered saline (PBS) solution at a concentration of 2%. The solution was sterilized by passing through 0.45- and 0.22-µm filter papers, respectively. Calcium chloride was dissolved in sterile-distilled water to prepare a Ca2 + solution at a concentration of 50 mM, and the solution was passed through sterile 0.22-µm filter paper. Second, tumor cells, cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco/Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO2 and 95% air, were harvested from flasks with 0.25% trypsin-EDTA and rinsed twice in PBS. Third, a dense cell pellet, prepared via centrifugation at 313× g for 10 min (2000 rpm in a IEC Centra-CL2 Benchtop centrifuge; International Equipment Company, Needham Heights, MA, USA), was mixed with the alginate solution at a volume ratio of 1:1. Fourth, Ca2 + solution was spread on a sterile glass slide to form a thin liquid film. Fifth, the mixture of tumor cells and alginate solution was loaded into the syringe and then extruded through a 30-gauge needle into the calcium solution. A disk-shape film was formed rapidly, due to the reaction between alginate and Ca2 + . Its diameter was approximately 2 mm, and the number of tumor cells per disk was approximately 2 × 105. The alginate film containing tumor cells was then incubated in the Ca2 + solution for 1 min, followed by incubation in PBS for 1 min. At the end of the PBS incubation, the film was gently transferred onto the center of the dorsal skinfold chamber using a needle tip. Finally, the chamber was sealed with a sterile glass coverslip.Immediately after the implantation, the cell pellet could be observed clearly at the center of the dorsal skinfold chamber. At approximately 1 h after the animals woke up from anesthesia, the pellet started to disappear from the implantation site. At 5 days postimplantation, tumor cells spread randomly and caused hemorrhage in the chamber (Figure 1A). The spreading problem could not be solved by increasing the density of cell pellets through increasing the centrifugation speed and period (data not shown). In contrast, tumor cells encapsulated in the alginate film did not spread. They could induce angiogenesis and eventually produce a solid tumor in the chamber (Figure 1B). The success rate of tumor production was 100% in our study (n = 38). In a control experiment, we implanted empty alginate films in the chamber and did not observe angiogenesis at 5 days postimplantation (Figure 1C).Figure 1. Images of dorsal skinfold chambers at 5 days after implantation of (A) a dense pellet of 4T1 cells, (B) 4T1 cells encapsulated in the alginate film, and (C) an empty alginate film.The images were taken using a Sony DSC-P1 digital photo camera (Sony, Tokyo, Japan) purchased from an electronic store.Figure 2 shows a sequence of images of tumor growth in the chamber after the implantation of tumor cells encapsulated in the alginate film. At day after implantation, the film was transparent and located at the center of the chamber (Figure 2A). On days 3 and 5, we observed dilated and tortuous microvessels as well as new capillary sprouts near the edge of the alginate film (Figure 2, B and C). However, angiogenesis was minimal in areas far away from the film, indicating that there was no spreading of tumor cells. On day 5, the alginate film became opaque, compared with that on day 1, presumably due to an increase in cell density and extracellular matrix secreted by the tumor and penetrated host cells. From day 5 to day 10, more microvessels were formed in the tumor, and the tumor grew bigger (Figure 2, C–F). Microcirculation in the tumor could be observed on day 8, indicating that the newly formed microvessels became functional at this stage.Figure 2. Images of a tumor growing in a dorsal skinfold chamber at different time points postimplantation of 4T1 cells encapsulated in the alginate film.(A) Day 1, (B) day 3, (C) day 5, (D) day 8, (E and F) day 10. Scale bars, 1 mm. The images were taken under an Axioscope™ light microscope (Carl Zeiss, Thornwood, NY, USA) using a Model ZVS-3C75DE color video camera (Carl Zeiss) and digitized using a Scion Frame Grabber and its image analysis software (Scion, Frederick, MD, USA).In addition to 4T1 cells, we implanted a human pharyngeal squamous carcinoma cell line (FaDu) into dorsal skinfold chambers in nude mice, using the same method discussed above. The results in terms of tumor production and growth were similar to those in 4T1 tumor studies (data not shown), indicating that the method was cell line independent.Comparing to tissue fragment implantation, the advantage of the new method is 4-fold. First, it can reduce variations in experimental observations caused by not being able to control the number of viable tumor cells in a tissue fragment. Second, the success rate of tumor production from the cells in the alginate film is 100% in our study, which is significantly higher than that from a tissue fragment (though highly variable, we have found that these assays have an average success rate of <50%; data not shown). Third, it is relatively less expensive and more reproducible to maintain tumor sources in a culture medium than in animals. Fourth, it reduces the use of animals in cancer research. The main disadvantage of the new method is that the initial growth of tumors occurs in an alginate gel instead of a piece of tissue. However, the microenvironment of tumor cells will improve rapidly after the tumor starts to grow, since the tumor and stromal cells that penetrate into the tumor will release extracellular matrix by themselves (17–20).In conclusion, we developed a novel and simple method to implant tumor cells in the dorsal skinfold chamber. 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CA94019.Competing Interests StatementThe authors declare no competing interests.PDF download
Source: BioTechniques
DOI: 10.2144/000112082
Authors: Yong Wang, Qing Chen, Fan Yuan
Institution: Duke University
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $1,415
OpenAlex ID: W2000053068