Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

Abstract

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated highfidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type–dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis

“…Spheroids can be removed directly from the bottom of the drop using a pipette aspirating a volume of 2-3 ml. A parallel spheroid transfer can be achieved by bringing all drops simultaneously into contact with a flat plate or with the wells of 384-well plate, similar to methods presented by Cavnar et al 47 We easily could recover all spheroids from 16-24 drops without any loss of viability.…”

Biomek Cell Workstation: A Flexible System for Automated 3D Cell Cultivation

“…12 Spheroid cultures are primarily formed from cancer cell lines: human hepatoma cell line HepG2 (ATCC HB-8065), MCF-7 (ATCC HTB-22), 15 cell lung cancer (NSCLC) cell lines (A549, Colo699), 16 MDA-MB-231 breast cancer cells, HeyA8 ovarian cancer cells, and human mammary green fluorescent protein (GFP)-fibroblast cell line. 17 Furthermore, we also manufactured 3D constructs in the form of spheroid cultures with cervix carcinoma cells to produce in vitro tumors without carrier material. Spheroids can also be formed by different cell lines, such as African green monkey kidney fibroblast cells (COS7), murine embryonic stem (mES) cells (ES-D3), and human epithelial carcinoma cells (A431.H9).…”

“…15,16 The alginate bead manufacturing is realized manually in six-well plates 13 or mainly in petri dishes by semiautomated formation processes. 17,[19][20][21] Regarding this, the cell detachment processes were manually performed followed by the automated droplet formation. Spheroids are self-aggregated clusters by domination of cell-cell interactions.…”

“…The semiautomated formation in 96-well plates displayed the basis for high-throughput manufacturing of pellet cultures. 19 Basically, the cells were manually detached and • Alginate hydrogel 23 • None [15][16][17][18] • None…”

“…• Petri dish 12,[23][24][25][26] • 384-well hanging drop plates 17,18 • 60 wells of a MicroWell MiniTray (Nunc) 15 • Gravity-PLUS microtissue culture system (InSphero AG) 16 • 15 mL tubes…”