Application of laser scanning imaging system in rapid detection and analysis of 3D tumor sphere or stem cell cloning sphere

Research on new anticancer drugs is currently facing significant challenges, as only 5% of the compounds that showed efficacy in preclinical studies continue to be developed and become licensed drugs. Traditional 2D cell culture models are used to evaluate drug candidates in the early stages of the drug discovery process. However, there is increasing evidence that cells grown in two-dimensional monolayers do not accurately reflect the biological complexity of tumors. .

The need for better in vitro models compatible with high throughput screening has led to the development of 3D cell culture models, especially multicellular microspheres, which will retain many of the morphological and hereditary traits of tumors. In in vitro experiments, 3D culture models are widely considered to be more physiologically relevant models than 2D forms of culture systems. The 3D model more accurately reflects the complexity of the in vivo microenvironment and is used in many research areas such as oncology [1], hepatotoxicity [2], neurobiology [3], pancreatic research [4], Nephrology [5], and stem cell research [6]. These studies have subverted our understanding of cell behavior in in vitro culture and in vivo. However, due to the limitations of cell culture techniques, high-throughput screening with 3D culture systems has been very slow. Technical bottlenecks include experimental heterogeneity, low throughput, difficulty in automating, and costly.

Here, we describe cell sphere assays for 3D culture by two methods: 1) in ultra-low attachment plates; 2) on semi-solid agar. Both methods can be performed on 96 and 384 well microplate formats.

We then used Acumen hci laser scanning technology for fast full-plate image acquisition (5 minutes/block), giving a range of parameters such as the number, area and volume of spheres. Acumen hci is a perfect system for high-content analysis of microspheres. Its unique full-hole scanning capability captures information on all microspheres at different locations in a single well, while a scanning mirror with a certain depth of field makes it possible to The spheres in each layer are also captured at one time, and the volume of a single sphere is counted without Z-layer tomography, and data is collected multiple times.

method:

A: Formation of tumor microspheres in ultra-low adhesion plates

Corining's ultra-low adhesion panels feature opaque walls and a clear, rounded bottom. A proprietary ultra-low cell adhesion coating with hydrophilic, bio-inert and non-degradable properties by covalent attachment to the inner surface of the bottom of the well. This unique design on the bottom of the culture well allows 3D cell spheroid cultures to grow highly reproducibly. The opaque sidewalls and the proprietary bottom mesh design greatly reduce the crosstalk between the fluorescent, luminescent background and the holes and holes. In the well, the spheroids can be generated, cultured, and detected for fluorescent or luminescent signals without the need to transfer the spheroids. Currently, Corning ultra-low adhesion sheets are available in 96- and 384-well plates, both of which are easy to automate. a) Single cells of hepatoma cell line HepG2 suspended in complete medium were plated in Corning ultra-low adhesion plate (#4520&3830), plated density was 96-well plate, 200 μl per well contained 300 cells, each in 384-well plate 50μl of well contains 300 cells b) Within 24 hours, the suspended cells will assemble into a single 3D microsphere and precipitate in the center of the cell, as shown in Figure 1A on the right. c) After 72 hours, replace 50% of the medium with fresh medium containing 2 times the concentration of Doxorubicin (doxorubicin). The final concentration after replacement is 316pM~3.16μM. d) The formed microspheres continue to culture for 6 days

Figure 1A

B: Formation of tumor microspheres in soft agar

C: staining and image acquisition

a) Add the dye Calcein-AM (final concentration 5 μM) to the wells containing the microspheres of the cells to stain the tumor microspheres formed by the two methods, and incubate for 1 hour at 37 degrees after the dye is added. b) The board then uses Acumen for image acquisition (5 minutes for image acquisition and data output per board) c) The data is analyzed online and obtained while scanning the image. The resulting report contains information on the number, area and volume of the sphere, fluorescence intensity, etc.

Figure 1C

result:

Tumor microspheres grow in ultra-low adhesion plates

Tumor microspheres grow in soft agar

to sum up:

The cell population, different cell types and micro-tissue structures growing in the 3D culture system coordinate and interact with each other, simulating the real living environment of the living substance in the micro-environment of the body, providing high quality for the researchers to analyze and overcome the disease. Data information. We established two simple and stable methods for 3D culture of tumor microspheres in 96 and 384 microplates. Information on number, area, volume and fluorescence intensity was obtained by staining and image acquisition. The consistency of the drug dose-effect curve reflects the reliability and reproducibility of the two methods. These data indicate that the acumen hci laser scanning system is a high-throughput (5 min/plate) of tumor microspheres obtained based on two culture systems. An ideal platform for analysis, which has been widely used in oncology [7-9] and stem cell research in the past [10-12].

References:

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