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Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

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Dissection of subanatomical regions (marked with red dotted line or arrows) from fresh frozen mouse coronal brain sections (20 µm thickness) using CTAS v.4.1. A.Collection of the left hilus of the dentate gyrus (hDG). B. Dissection of granular cells (G), molecular layer (M), and white matter (W) from mouse cerebellum. Intact (C) and dissected (D) anterior commissure, anterior (ACA) and right piriform cortex (Rpir). E. Dissected (left) and intact (right) thalamic and hypothalamic areas including posterior thalamic nucleus (1), part of ventral posteromedial thalamic nucleus (2), ventromedial thalamic nucleus (3), dorsomedial hypothalamic nucleus (4) and arcuate hypothalamic nucleus (5). Homotopical intact areas are outlined with dashed red lines. Tissues were stained with Toluidine Blue. Scale bar  = 250 µm. DCU ID = 50 µm; vacuum pulse duration: 100 ms; F. Representative microdissection of middle molecular layer of the dentate gyrus (a) and cellular layer of the subiculum (b) from fresh frozen nonstained mouse brain sections (20 µm thickness). Abbreviations: CA1–CA1 area of hippocampus; Sub – subiculum; DG – dentate gyrus; hf – hippocampal fissure. Scale bar  = 100 µm.
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pone-0041564-g003: Dissection of subanatomical regions (marked with red dotted line or arrows) from fresh frozen mouse coronal brain sections (20 µm thickness) using CTAS v.4.1. A.Collection of the left hilus of the dentate gyrus (hDG). B. Dissection of granular cells (G), molecular layer (M), and white matter (W) from mouse cerebellum. Intact (C) and dissected (D) anterior commissure, anterior (ACA) and right piriform cortex (Rpir). E. Dissected (left) and intact (right) thalamic and hypothalamic areas including posterior thalamic nucleus (1), part of ventral posteromedial thalamic nucleus (2), ventromedial thalamic nucleus (3), dorsomedial hypothalamic nucleus (4) and arcuate hypothalamic nucleus (5). Homotopical intact areas are outlined with dashed red lines. Tissues were stained with Toluidine Blue. Scale bar  = 250 µm. DCU ID = 50 µm; vacuum pulse duration: 100 ms; F. Representative microdissection of middle molecular layer of the dentate gyrus (a) and cellular layer of the subiculum (b) from fresh frozen nonstained mouse brain sections (20 µm thickness). Abbreviations: CA1–CA1 area of hippocampus; Sub – subiculum; DG – dentate gyrus; hf – hippocampal fissure. Scale bar  = 100 µm.

Mentions: To demonstrate that CTAS is capable of collecting desired tissue regions and cell clusters we performed a series of microdissections using mouse and rat brain tissues of various preparation types and thickness. Brain tissue was selected as the most heterogeneous and anatomically complex tissue type in the mammalian organism. Using CTAS we performed microdissection of fresh frozen and sucrose treated mouse and rat brain tissues and demonstrated its feasibility to precisely dissect desired subanatomical regions and cell clusters. Various tissue section thicknesses were tested and some of the suggested parameters are summarized in Table 1. The optimal range of tissue thickness was found to be between 10 µm and 300 µm depending on the preparation type. As shown in Fig. 3 and 4, the different subanatomical parts of the mouse brain were precisely dissected, including the hilus of the dentate gyrus (Fig. 3A), molecular layer, granular layers and white matter of cerebellum (Fig. 3B), anterior commissure, anterior and right piriform cortex (Fig. 3C–D), thalamic and hypothalamic areas including posterior thalamic nucleus, part of ventral posteromedial thalamic nucleus, ventromedial thalamic nucleus, dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus (Fig. 3E). Dissections were also performed on unstained sucrose treated (Fig. 3F) and fresh frozen tissues (Fig. 4). We also dissected different pyramidal layers of posterior CA3, posterior and ventral pyramidal layers of the hippocampus, as well as a mixture of granular and inner molecular layers of the dentate gyrus and lateral entorhinal cortex, individual layers of the motor cortex and piriform cortex (not shown). Using CTAS, different brain subanatomical regions could be collected from both stained and unstained fresh frozen and sucrose treated tissues (Fig. 3 and 4).


Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Dissection of subanatomical regions (marked with red dotted line or arrows) from fresh frozen mouse coronal brain sections (20 µm thickness) using CTAS v.4.1. A.Collection of the left hilus of the dentate gyrus (hDG). B. Dissection of granular cells (G), molecular layer (M), and white matter (W) from mouse cerebellum. Intact (C) and dissected (D) anterior commissure, anterior (ACA) and right piriform cortex (Rpir). E. Dissected (left) and intact (right) thalamic and hypothalamic areas including posterior thalamic nucleus (1), part of ventral posteromedial thalamic nucleus (2), ventromedial thalamic nucleus (3), dorsomedial hypothalamic nucleus (4) and arcuate hypothalamic nucleus (5). Homotopical intact areas are outlined with dashed red lines. Tissues were stained with Toluidine Blue. Scale bar  = 250 µm. DCU ID = 50 µm; vacuum pulse duration: 100 ms; F. Representative microdissection of middle molecular layer of the dentate gyrus (a) and cellular layer of the subiculum (b) from fresh frozen nonstained mouse brain sections (20 µm thickness). Abbreviations: CA1–CA1 area of hippocampus; Sub – subiculum; DG – dentate gyrus; hf – hippocampal fissure. Scale bar  = 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404047&req=5

pone-0041564-g003: Dissection of subanatomical regions (marked with red dotted line or arrows) from fresh frozen mouse coronal brain sections (20 µm thickness) using CTAS v.4.1. A.Collection of the left hilus of the dentate gyrus (hDG). B. Dissection of granular cells (G), molecular layer (M), and white matter (W) from mouse cerebellum. Intact (C) and dissected (D) anterior commissure, anterior (ACA) and right piriform cortex (Rpir). E. Dissected (left) and intact (right) thalamic and hypothalamic areas including posterior thalamic nucleus (1), part of ventral posteromedial thalamic nucleus (2), ventromedial thalamic nucleus (3), dorsomedial hypothalamic nucleus (4) and arcuate hypothalamic nucleus (5). Homotopical intact areas are outlined with dashed red lines. Tissues were stained with Toluidine Blue. Scale bar  = 250 µm. DCU ID = 50 µm; vacuum pulse duration: 100 ms; F. Representative microdissection of middle molecular layer of the dentate gyrus (a) and cellular layer of the subiculum (b) from fresh frozen nonstained mouse brain sections (20 µm thickness). Abbreviations: CA1–CA1 area of hippocampus; Sub – subiculum; DG – dentate gyrus; hf – hippocampal fissure. Scale bar  = 100 µm.
Mentions: To demonstrate that CTAS is capable of collecting desired tissue regions and cell clusters we performed a series of microdissections using mouse and rat brain tissues of various preparation types and thickness. Brain tissue was selected as the most heterogeneous and anatomically complex tissue type in the mammalian organism. Using CTAS we performed microdissection of fresh frozen and sucrose treated mouse and rat brain tissues and demonstrated its feasibility to precisely dissect desired subanatomical regions and cell clusters. Various tissue section thicknesses were tested and some of the suggested parameters are summarized in Table 1. The optimal range of tissue thickness was found to be between 10 µm and 300 µm depending on the preparation type. As shown in Fig. 3 and 4, the different subanatomical parts of the mouse brain were precisely dissected, including the hilus of the dentate gyrus (Fig. 3A), molecular layer, granular layers and white matter of cerebellum (Fig. 3B), anterior commissure, anterior and right piriform cortex (Fig. 3C–D), thalamic and hypothalamic areas including posterior thalamic nucleus, part of ventral posteromedial thalamic nucleus, ventromedial thalamic nucleus, dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus (Fig. 3E). Dissections were also performed on unstained sucrose treated (Fig. 3F) and fresh frozen tissues (Fig. 4). We also dissected different pyramidal layers of posterior CA3, posterior and ventral pyramidal layers of the hippocampus, as well as a mixture of granular and inner molecular layers of the dentate gyrus and lateral entorhinal cortex, individual layers of the motor cortex and piriform cortex (not shown). Using CTAS, different brain subanatomical regions could be collected from both stained and unstained fresh frozen and sucrose treated tissues (Fig. 3 and 4).

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

Show MeSH
Related in: MedlinePlus