3D Microbeam Studies in vitro


	RARAF – 3D Microbeam Studies in vitro
	3D Tissue Imaging As radiation experiments have moved more towards tissue samples and small organisms, the necessity to image optical sections within 3-D tissue was a driving factor in developing a multiphoton microscope into the endstation of the RARAF microbeam. One RARAF user planned a microbeam irradiation of a 3-D construct derived from human umbilical vein endothelial cells (HUVEC), which in initial studies, were optically sectioned using our multiphoton system. Derived from the walls of the umbilical vein, HUVEC cells were seeded onto a collagen matrix, a disk approximately 200 µm thick by 4 mm in diameter. While these cells “tunneled” through the matrix, forming a 3D structure similar to the capillaries of the blood circulatory system, collagen was displaced by cell growth and was degraded by cell enzyme action. A series of optical sections through these tissue samples (z-stacks) were compiled as movies to effectively transport the observer through the tissue slice. Sample z-stack movies are shown on the left. In these movie, cell nuclei are imaged by YOYO-1 (green), cell cytoplasm imaged with autofluorescence (red), and collagen imaged by second harmonic generation (blue). Red was used to color the SHG signal and blue was used for the AF signal. In addition to using the nuclear stain YOYO-1, two non-stain imaging modes were used to capture the cytoplasm and the collagen matrix: autofluorescence and second harmonic generation. These two non-stain imaging modes require less sample preparation and allow structural imaging in 3D. With the capacity for optical sectioning, the multiphoton microscope at RARAF offers a mode for observing radiation-induced effects, such as foci formation, within tissue. 3D Tissue Systems The radiation-induced bystander effect is the phenomenon whereby cellular effects such as sister chromosome aberrations, apoptosis, or transformations are expressed in unirradiated neighboring cells near an irradiated cell or cells. The bystander effect is generally believed to be due to cell-to-cell communication. Since it is well known that an organism is composed of different cell types interacting as functional units to maintain normal tissue function, experimental models that maintain tissue-like intercellular cell signaling and 3-D structure are essential for proper understanding of the bystander effect. Our goal is to provide users with an in-vivo-like multicellular system with preserved 3-D tissue microarchitecture and microenvironment. In order to fulfill these requirements, yet maintain good reproducibility, we use novel artificial human tissue systems, which are commercially available (MatTek Corporation, Ashland, MA). Artificial tissues reconstruct the normal tissue microarchitecture and preserve the in-vivo differentiation pattern. They are mitotically and metabolically active and release the relevant cytokines. Cells in these tissues also demonstrate the presence of gap junctions. Artificial tissues are cultivated using an air-liquid interface tissue culture technique (Fig. 1A). Tissue is grown on a semi-permeable membrane and fed with a serum-free medium from below (Fig. 1B). The human skin artificial tissue systems are cultivated on Millicell-CM culture inserts (Millipore), using a 28 µm hydrophilic PTFE membrane. The surface of the tissue is exposed to the air, which stimulates differentiation. Artificial tissues consist of a few layers of cells. The diameter of the MatTek tissues is about 8 mm, and their lifetime is 2 to 3 weeks depending on the tissue type. Artificial tissues are very stable and allow a high degree of reproducibility. This is a crucial advantage for microbeam experiments, which require a high degree of precision. On the other hand this is a very flexible system because many parameters including the type of cells, degree of differentiation and size can be controlled. The system is also cost effective, compared to working with primary explants, which requires an animal house. We have used a few types of artificial tissues for microbeam bystander experiments. In this example we will concentrate on EpiDerm, which is a human artificial skin system. EpiDerm consists of normal, human epidermal keratinocytes, which have been cultured to form a multilayered, differentiated model of the human epidermis. It closely resembles human skin microarchitecture with in-vivo like morphological and growth characteristics, which are uniform and highly reproducible. We are using three modifications of EpiDerm: EPI-200-3s, EPI-201 and EPI-200. EPI-200-3s (Fig. 2A) is a non-differentiated model; it consists of 1-2 cell layers and is not more than 10 mm thick. EPI-201 (Fig. 2B) is an intermediately-differentiated model; it consists of 3-5 cell layers and is 20-45 mm thick. Finally, EPI-200 (Fig. 2C) is a differentiated model of the epidermis; it consists of 10-12 cell layers and is 75-100 mm thick. Various degrees of differentiation are important because we demonstrated the importance of differentiation as a major component of the bystander response in the urothelial explant model. On the other hand, both the EPI-200-3s and EPI-201 models, if cultured, eventually form a system similar to the fully differentiated EPI-200. This gives us the opportunity to irradiate the undifferentiated EPI-200-3s model and study delayed bystander effects 7 days later when a fully differentiated multilayer structure would be formed - a study on the bystander response in the progeny of irradiated cells. Morphologically, EpiDerm consists of basal, spinous, granular, and cornified layers analogous to those found in an in-vivo epidermis. The system is mitotically and metabolically active. Markers of mature epidermis-specific differentiation such as pro-filaggrin, K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase are expressed in the EpiDerm system. Analysis of the tissue microstucture has proved the presence of keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of an in-vivo epidermis. Microbeam Irradiation The Columbia University single-cell / single-particle microbeam was used for tissue irradiation. We microbeam irradiated each tissue sample in a known pattern, so that all the irradiated cells are in one plane. Ten a-particles (~7.2 MeV) per location were delivered every 20-100 µm along the irradiation plane. A tissue sample is 8 mm in diameter. The human skin artificial tissue system is cultivated on Millicell-CM culture inserts (Millipore), using a 28 mm hydrophilic PTFE membrane. The tissue samples are irradiated from below through the membrane that forms the base of the culture insert. After traversal of the membrane a 7.2 MeV a-particle beam would have a remaining energy of about 2 MeV. This is enough energy to traverse about 10-15 mm of tissue, a range sufficient to target the basal cellular layers of EPI-200 and EPI-201 and to traverse all layers of EPI-200-3s artificial tissues. In order to maintain high precision, the culture insert is positioned in a custom-designed holder (Fig. 3A) and held with a fixture (Fig. 3B) to maintain stability. The holder is positioned on the microbeam stage (Fig. 3C), and the tissue is irradiated from below. The full microbeam cell dish assembly is represented in Fig. 3D. It is covered with a fixture to prevent contamination and drying-up during irradiation. The irradiation is fully automated, with a user-friendly interface to specify the number of protons per irradiation point and the separation between the irradiation points along the line of irradiation. Typical irradiation times vary from 1 to 4 minutes per tissue. After microbeam irradiation, each tissue was returned to a multi-well dish filled with fresh medium and incubated at 37°C in a humidified atmosphere with 5% CO₂, prior to fixing for 3 days. Paraffin Histological Section Preparation After incubation tissues were fixed in 10% neutral buffered formalin, paraffin embedded, and cut in 5 mm segments along the X axis (see Fig. 4) to prepare histological sections. We used vertical sectioning to physically isolate tissue fragments which contained directly irradiated cells from other fragments containing cells which had not been irradiated but were at known distances from irradiated cells. Figure 4 demonstrates our approach in detail. Fig. 4. After incubation, tissues were fixed in 10% neutral buffered formalin, paraffin embedded, and cut in 5 mm segments along the X axis to prepare histological sections. Endpoints After cutting and mounting on slides, sections were stained for the endpoints of interest. We performed a routine haemotoxylin-eosin (H&E) staining for every series of slides to assess the tissue morphologically. The next step was a quantitative assessment of the apoptosis contribution to the bystander effect using a TUNEL enzymatic in-situ labeling kit (DermaTACS) optimized for paraffin sections (Figs. 5, 6). The fraction of apoptotic cells is counted and the spatial distribution recorded. We also studied bystander-induced differentiation under in-situ conditions using immunohistochemical markers such as pro-filaggrin, K1/K10 cytokeratin pair (data not shown). Positive apoptotic cells appear bright blue (TACS Blue Label) on the photograph below. The image in figure 5 was located 500-700 µm from the irradiated line. Figure 6 demonstrates that there was an increased fraction of apoptotic cells versus control in the EPI-200 human artificial tissues. The fraction of apoptotic cells was measured at different distances, ranging from 200 to 1100 mm, away from the line of irradiated cells (10 a-particles every 100 mm) on day 3 after irradiation. The average fraction of apoptosis for all layers in irradiated samples is 3.7±0.6% versus 1.3±0.3% for all layers in control samples. Importantly, there are no statistically significant variations in the expression of bystander apoptosis over the 900 mm distance. This suggests that the bystander response has a long range and is propagated through 3-D tissues, possibly mediated by gap junctions. Fig. 5. Bystander-induced apoptosis in the artificial human skin system EPI-200 stained with the DermaTACS apoptosis kit. Positive apoptotic cells appear blue. Formalin fixed, paraffin embedded 5 µm histological sections are shown as imaged through a transmission light microscope (600X). Fig. 6. Fraction of apoptotic cells (TUNEL assay) in unirradiated bystander cells at different distances from irradiated cells in 3-D human artificial skin tissue (EPI-200, 10 a-particles every 100 mm) on day 3 after irradiation. Error bars represent standard deviations of the means (SEM). Each point (and SEM) is derived from at least 3 repeat experiments. Statistical Analysis Each experiment was performed with at least three different tissues and repeated at least for 3 times. All individual tissues were number-coded and scored blindly. The mean and standard deviation were calculated for all endpoint-positive and control cases. Significance tests were performed using the Student’s t test. In all statistical analyses, P < 0.05 was considered as statistically significant. Results and Discussion In addition to the skin models already discussed, we have performed microbeam and broad-field experiments with two other human artificial tissue systems: a reconstruction of the tracheal/bronchial tissue of human respiratory tract EpiAirway (AIR-110) and a corneal model (OCL-200). Human artificial skin systems have been carefully characterized; we performed extensive morphometric measurements and tested cultivation procedures for these models. We have developed unique techniques for broad-field and microbeam irradiations of human artificial tissue systems. The nature of the bystander experiments also requires high precision in positioning and registration of the samples during and post-irradiation. We have designed, in collaboration with the Design & Instrument Shop of the Center for Radiological Research, a set of fixtures (Fig. 3), used for microbeam and broad-field irradiation as well as for cultivation and histological processing. In summary, we now have a well-tuned methodology for microbeam-based bystander experiments on 3-D tissue samples. Reference Belyakov O.V., Mitchell S.A., Parikh D., Randers-Pehrson G., Marino S.A., Amundson S.A., Geard C.R. and Brenner D.J. Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away. Proc Natl Acad Sci U S A 102:14203-14208 (2005). [abstract] [PDF]
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