3D Cell Culture Support
- Q. Are the micro-molds that are used to cast 3D Petri Dishes® reusable and can they be autoclaved?
- A. Yes, the micro-molds are reusable up to 12 times and can be sterilized via a standard steam autoclave (30 min, dry cycle).
- Q. How should I store the micro-molds when not in use?
- A. The micro-molds should be stored in a covered container to maintain sterility and to avoid collecting dust or fibers on the small features.
- Q. Can I view my 3D microtissues with the microscope?
- A. Yes, the 3D Petri Dish® is cast from agarose so 3D microtissues can be easily viewed using standard inverted microscopes. Brightfield, phase contrast, fluorescent and time lapse images have been acquired of microtissues in the 3D Petri Dish®.
- Q. Do I have to seed by hand each of the small 256 recesses (Model 12-256) to form a microtissue in each of the 256 recesses?
- A. No, mono-dispersed cells are pipetted into the single large seeding chamber (190μl) of the 3D Petri Dish® directly above all the recesses. After this single pipetting step, the cells settle and partition into the 256 small recesses where they settle to the bottom of each recess and then form a microtissue. So with one pipetting step, 256 microtissues are formed. All models of the 3D Petri Dish® have a single seeding chamber above their recesses and so microtissues are formed in the same manner.
- Q. How do I control the size of my spheroids?
- A. When mono-dispersed cells are seeded into the cell seeding chamber of the 3D Petri Dish®, the cells settle, partition into each of the small recesses below the seeding chamber and then self assemble a microtissue at the bottom of each recess. Thus, spheroid size is controlled by the total number of cells seeded into the seeding chamber (i.e., more cells equals larger spheroids).
- Q. Can I be certain my cell type will form a 3D microtissue?
- A. Over 20 different cell types from a wide variety of tissues (mammalian cell lines and primary cells) have formed 3D microtissues in the 3D Petri Dish® and the list is growing. The rate and extent of microtissue formation varies with the adhesiveness and the cytoskeletal activity of each cell type. No guarantees at this point, but if a cell type is adhesive and is grown attached on a conventional flat plastic tissue culture dishes it will likely form 3D microtissues.
- Q. How fast will my cells self-assemble microtissues in the 3D Petri DishesTM?
- A. The rate of self-assembly varies with the adhesiveness and cytoskeletal activity of each cell type. The experience so far is 12 hours to 3 days.
- Q. How will I know when self-assembly is complete for my microtissue?
- A. The rate of self-assembly varies with each cell type (~ 12 to 72 hrs). During self assembly, the dimensions of the microtissue changes as the cells aggregate. For a spheroid, the x, y radii decrease with time and these measurements can be used to assess formation of the microtissue.
- Q. Can I assume that each spheroid is a perfect sphere?
- A. No, it’s well known from published scientific literature that multi-cellular spheroids are not always perfect spheres. Depending on cell type, some multi-cellular spheroids will approximate a sphere with equivalent x, y, z radii, whereas others will approximate an oblate spheroid geometry with equivalent x, y radii, but a shorter z radius.
- Q. Can I form mixed microtissues with two different cell types?
- A. Yes, depending on the cell types. Mixed microtissues have been formed from a mixture of 2 and even 3 cell types. Prior to seeding the 3D Petri Dish® , each cell type is trypsinized, counted and mixed with the other cell types at the desired ratio. This mixture of mono-dispersed cells is then seeded into the cell seeding chamber of the 3D Petri DishesTM. The cell mix settles, partitions into the recesses and then self-assembles a mixed microtissue at the bottom of recess.
- Q. If I make mixed microtissues from 2 different cell types what will be the location of each cell type in the mixed microtissue?
- A. The location of two different cell types within a mixed spheroid varies with cell types. In some cases, cells will self-sort, with one cell type forming the core and the other cell type forming the outer shell. In other cases, two cell types will be randomly distributed within the spheroid. Other patterns of cell mixes have also been observed. The sorting pattern of an untested pair of cells should be determined experimentally.
- Q. Will my cells proliferate in 3D?
- A. This depends on the cell type. Some cell types will proliferate rapidly in this 3D environment, whereas other cell types show little to no proliferation after formation of a microtissue.
- Q. Can I form clonal spheroids from individual cells?
- A. Yes, if the cell type proliferates in this 3D environment, it may be possible to form clonal spheroids. Try seeding the 3D Petri Dish® with very low cell numbers (< 1 cell per recess). The recesses are arranged in an array, so it’s possible to follow growth in specific recesses over time.
- Q. Can I perform Western blots and RT-PCR on microtissues formed in the 3D Petri Dish®?
- A. Yes, microtissues can be harvested from the 3D Petri Dish® and have been used for Western blots and RT-PCR.
- Q. My cells grow in a special medium, can I use it with the 3D Petri Dish®?
- A. The 3D Petri Dish® is cast from agarose, a hydrogel with high porosity that is readily equilibrated with a variety of cell culture media.
- Q. We don't have a centrifuge that handles multi-well plates, is there another way to harvest spheroids?
- A. Centrifugation is the easiest and most efficient method to harvest spheroids. Alternatively, it is possible to manually dislodge the spheroids. Invert the 3D Petri Dish® into a new tissue culture plate containing a very small amount of medium. Be careful to not create air bubbles between the upside down 3D Petri Dish® and the bottom of the new culture plate. Use a wide sterile spatula to smack the upside down 3D Petri Dish® to dislodge the spheroids. View the inverted 3D Petri Dish® under the microscope before and after the procedure to ensure that the spheroids have been released.
- Brown University, Providence, RI
- University of Virginia, Charlottesville, VA
- University of California San Francisco, San Francisco, CA
- Women & Infants Hospital, Providence, RI
- Rhode Island Hospital, Providence, RI
- University of Connecticut, Storrs, CT
- University of Texas, MD Anderson Cancer Center, Houston, TX
- Harvard Medical School, Boston MA
- VT-WFU School of Biomedical Engineering and Sciences, Blacksburg, VA
- Georgia Institute of Technology, Atlanta, GA
- University of Edinburgh, Edinburgh, Scotland
- Imperial College, London, UK
- CIC bioGUNE, Bizkaia, Spain
- University of Ghent, Ghent, Belgium
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
- University of York, Heslington, York, UK
- Duke University
- Columbia University
- University of Hong Kong
- University of Central Florida
- Clemson University
- University of Nottingham, UK
- Medical University of South Carolina
- University of Colorado
- Marine Biology Labs
- Bar-llan University
- IST, Austria
- Desroches BR, Zhang P, Choi BR, King ME, Maldonado AE, Li W, Rago A, Liu G, Nath N, Hartmann KM, Yang B, Koren G, Morgan JR, Mende U. Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells.
- Napolitano, A.P., Chai, P., Dean, D.M., Morgan, J.R. Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micro-Molded Non-Adhesive Hydrogels. Tissue Engineering, 13(8): 2087-2094. 2007.
- Dean, D.M., Napolitano, A.P., Youssef, J., Morgan, J.R. Rods, Toroids and Honeycombs. The Directed Self-Assembly of Microtissues with Prescribed Microscale Geometries. FASEB J., 21:4005-4012, 2007.
- Napolitano, A.P., Dean, D.M., Man, A.J., Youssef, J., Ho D.N., Rago, A.P, Lech, M.P., Morgan, J.R. Scaffold-free 3-Dimensional Cell Culture Utilizing Micro-Molded Non-Adhesive Hydrogels. BioTechniques, 43: 494-500, 2007.
- Barbone, D., Yang, T-M, Morgan, J.R., Gaudino, G., Broaddus, V.C. mTOR Contributes to the Acquired Multicellular Apoptotic Resistance of Human Malignant Mesothelioma Spheroids. J. Biol. Chem. 283, 13021-13030, 2008.
- Dean, D. M. and Morgan, J.R. Cytoskeletal-Mediated Tension Modulates the Directed Self-assembly of Microtissues. Tissue Eng. 14, 1989-1997, 2008.
- Rago, A.P., Chai, P., Morgan, J.R. Encapsulated Arrays of Self-Assembled Micro-tissues: An Alternative to Spherical Microcapsules. Tissue Eng. 15: 387-395, 2009.
- Rago, A.P., Dean, D. M., Morgan, J.R. Controlling Cell Position in Complex Heterotypic 3D Microtissues by Tissue Fusion. Biotech. and Bioeng. 102, 1231-1241, 2009.
- Dean, D. M. and Morgan, J.R. Fibroblast Elongation and Dendritic Extensions in Constrained Versus Unconstrained Microtissues. Cell Motility and the Cytoskeleton 66: 129-141, 2009.
- Livoti, C.M., and Morgan, J.R. Self-Assembly and Tissue Fusion of Toroid-Shaped Minimal Building Units. Tissue Eng. 16: 2051-2061, 2010.
- Krotz, S.F., Robins, J.C., Ferruccio, T-M., Moore, R., Steinhoff, M.M., Morgan, J.R. and Carson, S. In Vitro Maturation of Oocytes via Pre-fabricated Self-assembled Artificial Human Ovary. J. of Assisted Reproduction and Genetics. In press and online.
- Bao, B., Jiang, J. Yanase, T., Nishi, Y., and Morgan, J.R. Connexon-mediated Cell Adhesion Drives Microtissue Self-assembly. FASEB J. 25: 255-264, 2011. In press and online.
- Robins, J.C., Morgan, J.R., Krueger, P. Carson, S.A. BioengineeringAnembryonic Human Trophoblast Vesicles. Reproductive Sciences 18:18: 128-135, 2011.
- Youssef, J., Nurse, A., Freund, L.B. and Morgan, J.R. Quantification of the Forces Driving Self-assembly of 3D Micro-tissues. Proc. Nat’l. Acad. Sci. USA. 108: 6993-6998, 2011.
- Brian Bao, Charles P. Lai, Christian C. Naus, Jeffrey R. Morgan Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton ASBMB 2012.
- Klopper, A.V., Krens, G., Grill, S.W. and Heisenberg, C.-P. Finite-size Corrections to Scaling Behavior in Sorted Cell Aggregates. European Physical Journal E, 33: 99-103, 2010.
- Rago, A.P., Napolitano, A.P., Morgan, J.R. Miniaturization of an Anoikis Assay Using Non-Adhesive Micromolded Hydrogels. Cytotechnology, 56: 81-90, 2008.
- Yang, T-M., Barbone, D., Fennell, D.A., Broaddus, V.C. Bcl-2 Family Proteins Contribute to Apoptotic Resistance in Lung Cancer Multicellular Spheroids. Am. J. Respir. Cell. Mol. Bio. 41: 14-23, 2009.
- Kunstar, A., Otto, C., Karperien, M., van Blitterswijk, and van Apeldoorn, A. Raman Microspectroscopy: A Noninvasive Analysis Tool for Monitoring of Collagen-Containing Extracellular Matrix Formation in a Medium-Throughput Culture System Tissue Engineering 17: 737-744, 2011.
- Gwyther, T.A., Hu, J.Z., Christakis, A.G., Skorinko, J.K., Shaw, S.M., Billiar, K.L., Rolle, M.W. Engineered Vascular Tissue Fabricated from Aggregated Smooth Muscle. Cells. Cells, Tissues Organs 194:13-24, 2011.
- Mironov,V., Kasyanov, V., and Markwald, R.R. Organ Printing: From Bioprinter to Organ Biofabrication Line. Current Opinion in Biotechnology In Press.
- Elbert, D.L. Bottoms-up Tissue Engineering. Current Opinion in Biotechnology In Press.
- Boutin ME, Hoffman-Kim D. Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres.
–Human dermal fibroblasts
- Cardiac cells
–Rat cardiac myocytes
–Rat cardiac fibroblasts
- Endothelial cells
- Breast cancer cell lines
–Human: MDA-MB-231 cells
- Other epithelial cells
- Liver cell lines
- Reproductive system
–Human: Granulosa cells
–Human: TCL trophoblast cells
–Human: Theca cells
- Neuronal cell lines
–Rat: RG2, neuroblastoma
–Rat: 9L, glioma
–Rat: A7; astrocytes
-Murine: B104 neuroblastoma
- Zebrafish ectoderm progenitor cells
- Zebrafish mesoderm progenitor cells
- Human non-small cell lung cancer cells, A549 cells
- Human non-small cell lung cancer cells, H1299
- Primary bovine chondrocytes
- Rat aortic smooth muscle cells
www.allelefrequencies.net . An online repository that contains information on the frequencies of immune genes and their corresponding alleles in different populations. Nucleic Acid Research 2011, 39, D913-D919.