Цыпленок Ех ово Культура и Ех ово CAM Пробирной: как это действительно работает
Summary
Куриных chorioallantoic мембраны (CAM) является уникальным, естественно иммунодефицитных благоприятные условия для изучения культуры ангиогенеза и опухолей. Это видео статья демонстрирует различные этапы на куриных<em> Ех ово</em> Культура, применение потенциально ангиогенных веществ и успешной прививки опухолевых клеток и тканей на поверхности CAM.
Abstract
Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.
Protocol
Discussion
Инновационные или просто еще один протокол куриных культуры?
С бывшего корпуса менее протоколы культуры 1-4 общие показатели выживаемости куриных эмбрионов были низкими, например, ок. 30% 1. Изысканный бывший ово протокол культуры, описанные в этой статье ?…
Acknowledgements
Авторы хотели бы поблагодарить Дж. Kueper и Дж. Wittschier за отличную техническую помощь и проф Ruebben для снабжать опухоль материала.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fertilized Eggs | Animal | Söries Trockels, Germany | ||
| Mice | Animal | Charles Rivers Laboratories | ||
| Incubator Type 3000 with rotating egg tray | Tools | Siepmann GmbH Germany | 9503 | Maintain at 37°C with relative humidity set above 60% |
| Egg incubator BSS 160 | Tools | Grumbach, Germany | 8101 | Maintain at 37-387deg;C with relative humidity set above 60% |
| PBS | Reagent | Sigma | PBS should be cold (> 4°C) and sterile | |
| Dulbecco’s modified eagle’s medium | Reagent | Sigma | D 6429 | DMEM culture medium |
| Leibovitz’s L-15 Medium | Reagent | Invitrogen | 31415-029 | here: collection medium for tumor samples |
| Tumor cell -specific medium | Reagent | Sigma | each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz’s Medium or Dulbecco’s modified eagle’s medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 μM insulin |
|
| 70% EtOH | Reagent | Sigma | ||
| Magnetic stir bar, triangled 80 mm | Tool | VWR | 442-0391 | A triangled magnetic stir bar serves well to crack the eggs shell. |
| Forceps DUMONT #5 | Tool | Fine Science Tools | 11252-30 | bevelled very fine shanks (0.05 mm x 0.02 mm tip) |
| Forceps DUMONT #7 | Tool | Fine Science Tools | 11271-30 | curved shanks (0.07 mm x 0.10 mm tip) |
| Spring scissors, straight, 8cm | Tool | Fine Science Tools | 15000-00 | fine, small straight blades |
| Fine Iris scissors, straight | Tool | Fine Science Tools | 14094-11 | Use to cut our the CAM |
| Standard scissors, straight, sharp/blunt | Tool | Fine Science Tools | 14007-14 | Use for decapitation or cervical dislocation |
| microliter syringe 1702 TLLX, 25 μl | Tool | Hamilton | 80222 | Use for injection into the vitreous |
| Hamilton 33-G needle (15 mm) | Tool | Fine Science Tools | 18073-15 | Use for injection into the vitreous |
| Sterile scalpels | Tool | Use for dissecting tumor samples | ||
| Small drain spoon | Tool | Geuder, Germany | 15758 | Use to transfer of small chick embryos |
| Wax board with fixing pins | Tool | Self made | Use to fix of animals for dissection | |
| Petri dishes 60 x 15 mm | Tool | Greiner | 628102 | |
| Petri dishes 92 x 16 mm | Tool | Sarstedt | 82.1472 | |
| Sterile Petri dish 100 x 20 mm | Tool | Greiner | 664160 | extra deep, with spacers for ventilation |
| Beaker | Tool | 300-600 ml | ||
| FALCON tubes | Tool | FALCON | 15 ml and 50 ml | |
| Eppendorf tubes | Tool | Eppendorf | 1.5 ml and 2 ml | |
| 1ml pipette tips | Tool | Use to cut plastic rings for application of substances / cells | ||
| Peripheral venous catheter (PVL) | Tool | Viggo® | Use to cut silicon rings from the tube for application of substances / cells | |
| Pipettes and tips | Tool | Eppendorf / Abimed | ||
| Autoclaved folded Filters 595 1/2, 110 mm diameter | Tool | Schleicher & Schuell | 311643 | Carrier, which caused least irritation of the CAM; used in the paper |
| PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm | Tool | santec-medical | REF 277, LOT 832/511-1 | Carrier; caused false positive results |
| Hxdroxyethylcellulose Tylose H 100000 | Reagent | Carrier mixed with Kollidon 17 PF; caused false positive results | ||
| Kollidon 17 PF | Reagent | BASF | S30200 | mixed with Hydroxyethylcellulose; caused false positive results |
| Collagen Biomatrix TissuDura | Tool | Baxter Healthcare S.A. | REF B2246000999999 | Carrier; caused false positive results |
| Round glass cover slips, 11 mm | Tool | Assistent Germany | 1001/0011 | Carrier; causes false positive results |
| Bovine Collagen Sponge | Tool | Wyeth | Carrier; caused false positive results | |
| Resodont Absorbable Collagen Membrane | Tool | Resorba Woundcare Germany | LOT 280303 | Carrier; caused false positive results |
| Non-Woven Swabs | Tool | Fink & Walter GmbH | REF 328132 | Carrier; caused false positive results |
Referências
- Auerbach, R., Kubai, L., Knighton, D., Folkman, J. A simple procedure for long-term cultivation of chicken embryos. Dev. Biol. 41, 391-394 (1974).
- Tufan, A. C., Akdogan, I., Adiguzel, E. Shell-less culture of the chick embryo as a model system in the study of development neurobiology. Neuroanatomy. 3, 8-11 (2004).
- Deryugina, E. I., Quigley, J. P. Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules. Methods Enzymology. 444, 21-44 (2008).
- Ribatti, D. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis. Int. Rev. Cell Mol. Biol. 270, 181-224 (2008).
- Höckel, M., Sasse, J., Wissler, J. H. Purified monocyte-derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and “tube formation” but not proliferation of capillary endothelial cells in vitro. J. Cell Physiol. 133, 1-13 (1987).
- Wissler, J. H. Engineering of blood vessel patterns by Angio Morphogens [Angiotropins]. Materialwiss. Werkstofftech. 32, 984-1008 (2001).
- Kistler, H. B. . J. r. .., LaVail, J. H. Penetration of horseradish peroxidase into the optic nerve after vitreal or vascular injections in the developing chick. Invest Ophthalmol. Vis. Sci. 20, 705-716 (1981).
- Halfter, W. Change in embryonic eye size and retinal cell proliferation following intravitreal injection of glycosaminoglycans. Invest. Ophthalmol. Vis. Sci. 49, 3289-3298 (2008).
- Oppitz, M., Busch, C., Shriek, G., Metzger, M., Just, L., Drews, U. Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo. Melanoma Res. 17, 17-30 (2007).
- Uchibayashi, T., Egawa, M., Nakajima, K., Hisazumi, H., Tanaka, M., Endo, Y., Sasaki, T. Responses of tumour cell lines implanted onto the chorioallantoic membrane of chick embryo to anticancer agents in combination with hyperthermia. Urol. Res. 20, 237-239 (1992).
- Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane. Pathol. Oncol. Res. , (2008).
- Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. E. Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model. J. Urol. 166, 1502-1507 (2001).
- Hall, B. K. Grafting of organs and tissues to the chorioallantoic membrane of the embryonic chick. TCA Manual. 4 (3), 881-883 (1978).
- Kunzi-Rapp, K., Kaskel, P., Steiner, R., Peter, R. U., Krahn, G. Increased blood levels of human S100 in melanoma chick embryo xenografts’ circulation. Pigment Cell Res. 14, 9-13 (2001).
- McFall, R. C., Sery, T. W., Makadon, M. Characterization of a new continuous cell line derived from a human retinoblastoma. Pesquisa do Câncer. 37, 1003-1010 (1977).
- Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination. Pesquisa do Câncer. 65, 10959-10969 (2005).
- Kim, J., Kovalski, K., Ossowski, L. requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell. 94, 353-362 (1998).
