While, historically, 2D cultures have been the mainstay of in vitro assays, there is a developing interest in transferring to 3D models in an effort to increase the biological relevance of the measurement. By more closely reﬂecting conditions within the tissue, the hope is that such 3D models will help bridge the gap between in vitro and in vivo measurements thereby increasing the usefulness of the in vitro assay. Of equal importance to the aim of increasing biological relevance is the parameter measured. One of the most informative is to measure cell metabolism, whereby perturbed metabolism or mitochondrial function is probed without disrupting the 3D structure. This can be achieved in microtitre plate format using the MitoXpress Xtra HS and pH-Xtra products from Agilent Technologies.
MitoXpress Xtra HS measures oxygen consumption and therefore informs speciﬁcally on mitochondrial function, while pH-Xtra measures extracellular acidiﬁ cation and is therefore a convenient measure of glycolytic ﬂux. Here we demonstrate the application of these probe technologies to 3D cultures generated using the RAFT™ system from TAP Biosystems. RAFT™ facilitates the convenient production of consistent collagenbased structures. This, in conjunction with MitoXpress Xtra HS and pH-Xtra facilitates detailed microplate-based measurements of metabolic activity of 3D cultures without disrupting the integrity of the 3D structure. Measurements are conducted on the FLUOstar Omega microplate reader from BMG LABTECH.
Materials & Methods
- 3D RAFTTM cultures including black walled clear bottom 96-well cell culture microplates, MitoXpress Xtra-Oxygen Consumption Assay [HS Method], (Agilent Technologies, MX-200-4)
- pH-Xtra Glycolysis Assay, (Agilent Technologies, PH-200-4)
- FLUOstar® Omega microplate reader (BMG LABTECH)
- DMEM and culture media were obtained through usual distribution channels
3D RAFT cultures were prepared with either A549 or HepG2 cells (data not shown) at the indicated density in 240 μl DMEM/Collagen solution on a 96-well plate. RAFT cultures were formed as per manufacturer’s protocol (Fig. 1).
(Fig. 1A) Cells and neutralized collagen were mixed and pipetted into one well. After 15 min incubation at 37°C, a hydrogel is formed. Medium was absorbed from the hydrogel (Fig. 1B) to increase concentration of collagen and cells to in vivo conditions. The process is completed (Fig. 1C) in less than 1 hour and results in a structure that is about 120 μm thick.
Oxygen Consumption Measurements
Culture medium was removed after a desired culture period and 100 μl of MitoXpress Xtra stock solution prepared in prewarmed DMEM was added. 1 μl of compound (100x) was added to appropriate wells. All wells were then sealed by adding 100 μl prewarmed HS mineral oil to prevent the back diffusion of ambient oxygen. The plate was then measured kinetically on a FLUOstar Omega for 90-120 minutes at 37°C.
Extracellular Acidiﬁcation Measurements
Two hours prior to measurement the RAFT cell culture plate was placed in a CO2 free incubator at 37°C, 95% humidity, in order to remove CO2 from the plate material. Media was removed and 2 wash steps were performed using the Respiration Buffer (0.5 mM KH2PO4, 0.5 mM K2HPO4, 20 mM Glucose, 4.5 g/L NaCl, 4.0 g/L KCl, 0.097 g/L MgSO4, 0.265 g/L CaCl2). Finally, 150 μl of Respiration Buffer containing pH-XtraTM probe at the recommended concentration was added to each well. The plate was then measured kinetically.
FLUOstar Omega/CLARIOstar® instrument settings
|Measurement method:||Time-resolved fluorescence (Omega: TR-F Optical attachment installed)|
|Measurement mode:||Plate Mode Kinetic|
|Measurement time:||120 min (data points every 2 min)|
Dual chromatic using the following windows:
|Excitation/ Emission filters||Integration start/time (µs)|
1 TREx L + 655-50 (Omega) + 645-20 (CLARIOstar)
2 TREx L + 655-50 (Omega) + 645-20 (CLARIOstar)
pH-XtraTM Glycolysis Assay
1 TREx L and 615-BP10 100/40
2 TREx L and 615-BP10
Results & Discussion
Sample oxygen consumption profiles are presented in Figure 2.
Increasing the density of cells in the 3D matrix results in increased rates of oxygen consumption, with strong signal changes observed across the cell concentrations tested. Fig 3 shows the effect of treatment on cells within the 3D structure where ETC activity has been pharmacologically modulated. Sample acidiﬁcation proﬁles are presented in Figure 4 with signiﬁcant acidiﬁcation observed for untreated cells.
Acidiﬁcation is inhibited almost completely on treatment with oxamate indicating that the acidiﬁcation derives from the production of lactic acid. Treatment with antimycin however causes a signiﬁcant increase in acidiﬁcations the cell increases glycolytic ﬂux in order to maintain cellular ATP supply.
MitoXpress Xtra – Oxygen Consumption Assay and pH-Xtra Glycolysis Assay, provide a convenient, sensitive, and high throughput measure of mitochondrial function, metabolism, and cellular energy ﬂux when combined with the FLUOstar Omega multimode plate reader. These data illustrate the applicability of these assays to the study of cellular function in a complex multicellular 3D RAFTTM culture system without disrupting the integrity of the 3D structure. The FLUOstar Omega is enabled with menu selection for easy instrument set-up and data analysis, including the ability to input calibrations to generate O2 and H+ scales.