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ARCHITECTURE OF DORSAL TISSUE REAGGREGATES FROM XENOPUS LAEVIS EMBRYOS Uma Balakrishnan, Joseph Shawky, and Lance Davidson Department of Bioengineering, University of Pittsburgh DFA, the epithelium is also peeled off and discarded, as this INTRODUCTION layer takes longer to dissociate in the solution, which would be The central goal of tissue engineering is to recreate the unhealthy for the rest of the cells. form of clinically needed tissues together with their proper The dissociated cells are placed in a specially designed function. In tissue engineering today, 3D-printing is the newest microcentrifuge tube with 30 µL of agarose in a micropipette way to generate multicellular tissues for clinical use.[1] 3Dtip; the dissociation media is replaced with DFA. These cells printing relies on mixing scaffolds of extracellular matrix or are centrifuged for 10 minutes at 5 rpm (Fig 1c). This creates a hydrogels with cells as a starting material. This method of sheet of the non-differentiated cells to be cut into 3-4 building tissues can be useful today for tissues that have low reaggregates (Fig 1d). In those experiments that require fixation cell densities such as tendons or connective tissue; however, the after a specific amount of time, the reaggregates are placed majority of human tissues have a high ratio of cells to under glass and allowed to develop further. For each extracellular fluid.[2] Therefore, developing methods to engineer experiment, at least two additional dorsal isolates were tissues with high cell density is a critical problem that must be microsurgically removed to serve as controls. addressed for future clinical application. One such method to develop engineered tissues with a high cell density is to create tissue reaggregates.[2] In this method, dissociated, non-differentiated cells from a tissue are packed together using centrifugation, which mimics the forces present during development. Dissociation is required to break down any architecture present from the original tissue. Once centrifuged, the packed-together cells are a brick, called a reaggregate, of non-differentiated cells. The African clawed frog Xenopus laevis embryo makes a good model to develop this method to create scaffold-free engineered tissues because it develops outside the mother and can be microsurgically manipulated without damaging individual cells. Specifically, the dorsal tissue of X. laevis embryos between gastrulation and neurulation is a good tissue Figure 1 Creating a tissue reaggregate. A frog embryo is (a) to make aggregates because it contains all three germ layers dissected to remove a dorsal tissue. This tissue is (b) dissociated and (endoderm, mesoderm, and ectoderm) of undifferentiated (c) centrifuged to yield a (d) tissue aggregate. cells.[3] Having a model to make viable tissue aggregates is key The Live/Dead Assay identified cells with a compromised to developing high-density engineered tissues for clinical cell membrane to determine the viability of the aggregated application. tissue. Cells were stained at room temperature for 2 hours. For the other stains, the reaggregates were fixed in Dent’s OBJECTIVE & HYPOTHESIS Fix at 4 ºC overnight and stained using immunochemistry. The objective of this study is to evaluate various Anti-rabbit β-catenin primary antibody was used to tag characteristics of tissue reaggregates from X. laevis embryos. intercellular junctions. ZO-1 primary antibody was used to tag For this study to be successful, staining and imaging the tight junctions present in the epithelial layer. The fluorescent aggregates should give visual confirmation that the aggregates TRITC secondary antibody was used for all experiments. survived the reaggregation process, reformed cell-to-cell All Live/Dead and β-catenin staining occurred directly contacts, and grown an epithelium. after reaggregation. The ZO-1 staining happened at three METHODS distinct time points: (1) immediately after reaggregation, (2) X. laevis embryos are harvested, fertilized, allowed to age after six hours, and (3) after 24 hours. to stage 12.5-15, at least until blastopore is closed. Dorsal tissue All tissues were imaged using a confocal microscope. is microsurgically removed from the embryo using hair tools Images for the Live/Dead Assay and β-catenin staining are under a dissection microscope in Danilchik’s For Amy (DFA) cross-sections of the tissues. Images for ZO-1 staining are from solution with 0.1% bovine serum albumin (Fig 1a). In each the surface of the dorsal isolates and tissue aggregates. experiment, 10-12 dorsal isolates are explanted. Each explant is RESULTS dissociated in a Ca2+/Mg2+ free solution for 5-7 minutes to The Live/Dead Assay shows qualitatively that the majority cause cells to endocytose the intercellular junctions. of the cells are alive toward the core of the tissue (Fig 2a). On Additionally, the tissue is mechanically dissociated by the edges of the aggregate, there are many shriveled, dead cells chopping the tissue into individual cells (Fig 1b). Also while in 1 surrounding the aggregate (Fig 2b). However these cells do not appear as if they are attached to the actual tissue. Figure 2 Live/Dead Assay. Images from (a) the center and on (b) the periphery of a tissue aggregate with live (green) and dead (red) cells. Scale = 50 µm (n=7) The control tissue stained for β-catenin shows the highly specific and organized structure of cells in the dorsal tissue (Fig 3a). In the tissue aggregate, this architecture is completely disrupted, however there are clearly intercellular junctions present (Fig 3b). Figure 4 ZO-1 staining. Epithelium in (a) dorsal isolate and evidence of epithelial cells (white arrows) in tissue aggregates after (b) 0 hours (n=2), (c) 6 hours (n=3) and (d) 24 hours (n=2). Scale = 50 µm Figure 3 β-catenin staining. Intercellular junctions present in both the (a) dorsal isolate and (b) tissue aggregate (n=6). Scale bar = 50 µm ZO-1 staining revealed an overall lack of growth of epithelial cells on the surface of the tissue aggregates. Immediately after microsurgery, there is absolutely no fluorescent signal for ZO-1 on the surface of the aggregate as compared to the control epithelium on a dorsal isolate (Fig 4a,b). After 6 and 24 hours, some epithelium was present; however, it did not cover a significant portion of the tissue and its surface area did not seem to increase with time. DISCUSSION The qualitative data of this study strongly support that the tissue aggregates both survive the dissociation and reaggregation process and reform intercellular junctions with their new cellular neighbors. Although there are dead cells on the periphery of the tissue aggregates, the new tissue itself is viable and useful for future research. This study does not provide conclusive evidence that epithelial cells grow on the surface of the aggregate. The small numbers of cells present and lack of ZO-1 signal increase over time may suggest that these cells are simply left over from the original tissue, a result of incomplete discarding of the epithelial layer during dissociation. However, if more epithelium is shown to grow on the aggregate it may be evidence of trans-differentiation occurring at the surface where cells are exposed to different surface tensions than the cells on the inside of the tissue. This study could be further developed by comparing the volumes of dead and live cells to the tissue volume, quantifying disorganization in a tissue, and calculating the relative surface area which the epithelial cells cover on the aggregate surface. In the future, these aggregates can be induced to various factors to cause them to differentiate into various cell types. Multiple aggregates of differing cell types and shapes may be used to build larger structures, much like building a house using individual bricks. To use this aggregation method for clinical applications, aggregates must be made from human stem cells. However, first understanding and developing this method in a cheaper and easier to manipulate model is important to the eventual usefulness of tissue aggregates in tissue engineering. ACKNOWLEDGMENTS Thank you to the whole Davidson Mechanics and Morphogenesis Lab for their support on this project. REFERENCES 1. Cao, Y., Vacanti, J. P., Paige, K. T., Upton, J., & Vacanti, C. A. (1997). Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plastic and reconstructive surgery, 100(2), 297-302. 2. Miller, J. S. (2014). The billion cell construct: will threedimensional printing get us there? PLoS Biol, 12(6), e1001882. 3. Wilson, P. A., Oster, G., & Keller, R. (1989). Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants. Development, 105(1), 155166. 2