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Assessment of Chemosensitivity | Formation of Co-Culture Spheroids | Assay Performance

Versatile Formation of Co-Culture Spheroids using Perfecta3D™ Hanging Drop Culture Plates

The ability to co-culture and pattern multiple cell types in 3D can open up novel ways to study cancer biology, developmental biology, and tissue engineering and provide new approaches to understand and manipulate heterotypic cellular interactions in 3D. For example, co-culture of different colored cells allows for real-time tracking of cellular localizations, migration, self-organization, differentiation, and intercellular interactions in 3D.

Co-culture spheroids can be easily manipulated using the Perfecta3D™ Hanging Drop Culture Plates by varying the timing and order of seeding different cell types into the hanging drops. Perfecta3D™ Hanging Drop Culture Plates offer several advantages compared to conventional and alternative techniques using rotating bioreactors, non-adherent surfaces, magnetic particles, and round-bottom and V-bottom micro-wells in combination with centrifugation.

Perfecta3D™ Hanging Drop Culture Plates ensure uniform incorporation of all co-culture cell types into the spheroids. The culture system inherently offers the convenience of utilizing natural gravity force to aggregate all cell types into a single spheroid gently, making formation of mixed co-culture spheroids as simple as mono-culture spheroids. Unlike the use of centrifugation, which generates shear forces that can damage cells, or the use of magnetic particles, which can interfere with cellular behaviors and test compounds, the Perfecta3D™ platform offers a simple user-interface, free of external forces or substances.

Perfecta3D™ Hanging Drop Culture Plates have the unique advantage of lacking a bottom substrate for cells to eventually attach to over long-term cultures. The ability to access spheroid samples directly from the top of the plate not only greatly simplifies the steps required in manipulating co-culture spheroids, but also enable sophisticated patterning of spheroids that are not possible using other methods.

Mixed Co-Culture Spheroids

PC-3_DsRed human prostate cancer cells, human embilical vein endothelial (HUVEC) cells, and MC3T3-E1 mouse preosteoblast cells were seeded together at 1:50:50 ratio to form mixed co-culture spheroids. PC-3_DsRed cells, which were transfected with DsRed lentivirus to express red fluorescent protein, are randomly distributed within the spheroid.

Concentric Layer Patterning of Co-Culture Spheroids

Concentric layer patterning was achieved by initially forming a spheroid of one cell type as the inner core. Cell suspension of the second cell type was subsequently added to the existing hanging drop to form an exterior coating around the inner core.

PC-3_DsRed human prostate cancer cells and MC3T3-E1 mouse preosteoblast cells were co-cultured at 1:100 ratio, with PC-3_DsRed cells preferentially patterned in the center core of the spheroid, as the exterior coating of the spheroid, or randomly distributed within the spheroid.

CellTracker Green-labeled COS7 African green monkey kidney fibroblast cells were cultured to form spheroids. On day 3, CellTracker Red-labeled COS7 cells were added to each hanging drop already containing a CellTracker Green-labeled COS7 spheroid. The red cells attached to the periphery of the existing green spheroid and subsequently formed into a single spheroid with the green cells.

Janus Spheroids

Janus spheroids are spheroids composed of two groups of cells juxtaposed next to each other so that each group essentially forms a hemisphere. A Janus spheroid can be formed easily by retrieving a spheroid from the top of an access hole and then gently pipette into another existing hanging drop containing a spheroid, just like the removal and addition of liquid.

CellTracker Green-labeled and CellTracker Red-labeled COS7 African green monkey kidney fibroblast cells were cultured separately to form spheroids. On day 3, a green spheroid was transferred into each hanging drop containing a red spheroid. Over the next few days, the two spheroids slowly aggregated together to form a single Janus spheroid with half green cells and half red cells.

Images adapted from:
Tung et al. High-throughput 3D spheroid culture and drug testing using 384 hanging drop array. Analyst, 2011, 136, 473-478. > Read Paper
Hsiao et al. 384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids. Biotechnology and Bioengineering, 2011. > Read Paper