Three basic steps are involved in GMD assays: (i) forming GMDs from a cell suspension; (ii) carrying out assays within many GMDs simultaneously; and (iii) isolating microdrops of interest using physical methods such as fluorescence activated cell sorting (FACS).
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Micrographs of Gel Microdrops
(Click to see larger Image)
(Click to see larger Image)
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GMDs are composed of unique agarose-based materials (CelGel™ and CelBioGel™) and are prepared by dispersing the CelGel™ and CelBioGel™ (containing the cells of interest) into an excess of a non-aqueous phase (e.g. oil) to form an emulsion. The emulsion is transiently cooled causing the molten agarose drops (containing cells) to gel. Due to the thermal hysteresis property of agarose, the GMDs remain in the gel state even after the temperature is increased. Once gelled, the GMDs are physically distinct and can be physically manipulated in much the same way as one would manipulate cells (i.e. they can be centrifuged, pipetted, incubated,and sorted via flow cytometry).
GMD Size, Volume, and Permeability
Microdrop technology differs significantly from other gel entrapment technologies in that the particle size has been reduced from macroscopic proportions (>1 mm in diameter) to microscopic proportions (< 100 microns in diameter). The volume of GMDs produced by this process ranges from 1 nL (100 micron diameter) to 4 pL (20 micron diameter). GMDs are permeable to small molecules such as nutrients, ell culture media and antibodies. The pore size permits diffusion of molecules up to 900 kDa.
The size distribution of microdrops depends on the interplay of a number of key parameters. These include: the viscosity of the non-aqueous medium, the ratio of aqueous to non-aqueous fluid volumes, the amount of hydrodynamic shear applied (i.e. the rotation speed of the blade), and the presence of surface-active agents (i.e. surfactants such as detergents or lipids). Various dispersion methods produce a range of microdrop sizes. Analysis of GMDs using a Coulter multisizer particle analyzer demonstrates that GMDs produced using the CellSys 100™ Microdrop Maker and a well defined set of parameters have a narrow and reproducible distribution range, and majority of GMDs fall within a targeted size. For example. since mammalian cells are typically used for the secretion assay, a protocol in generating 35 µm GMDs are used. The requirement for preservation of cell viability for bioproccessing application places certain limitations on the degree to which some of the key variables can be manipulated.
Included with the CellSys 100™ Microdrop Maker are an ice water bath container and two 12.3 cm stainless steel shaft/blade assemblies. The CellSys 100™ Microdrop Maker is a sophisticated emulsifier designed to maximize cell encapsulation while preserving cell integrity. High rotation speeds and unique blade configuration allow the selection of microdrop diameter sizes between 10 and 200 microns.
Physical Dimensions:
Width: 20.3 cm
Depth: 25.4 cm
Height: 38.1 cmWeight: 6.8 kg
Power Input: 110-135 VAC, 50/60Hz, 1 amp
(European Spec. equipment is available)Rotational Speed Range: OFF to 2800 rpm
The CellSys 100™ microdrop maker is equipped with a moveable stage allowing immersion of the oil-agarose emulsion in an ice water bath while maintaining the proper shear force for uniform preparation of solidified gel microdrops. The CellSys 100™ microdrop maker's small footprint conserves laboratory bench space and facilitates transfer into a laminar flow hood.
CellSys 100™ Microdrop Maker
GMD Encapsulation Protocol
Harvest Cells
Mix cells with CelGel™ or CelBioGel™ and surfactants at 37°C.
Add agarose-cell suspension to emulsion matrix (oil phase).
Emulsify using CellSys 100™ microdrop maker
- 1 min. at 18-24°C, 2100 rpm
- 1 min. at 4°C, 2100 rpm
- 10 min. at 4°C, 1100 rpm
Remove GMDs from emulsion matrix by centrifugation
Wash with PBSEncapsulation takes 1 hour.
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Statistical Occupancy of GMDs
A basic property of GMDs formed with small numbers of initial cells per GMD is that Poisson statistics adequately describes the frequency-of-occurrence of initial cells within individual GMDs, as it does for microtiter well and petri dish inoculation. Thus, by knowing the mean diameter of GMDs produced under a given set of conditions, one can determine the total number of GMDs which will be generated. To obtain GMDs with a high probability of containing 0 or 1 initial cells, one would occupy only 10 to 20% of the GMDs, in a manner completely analogous to limited dilution cloning.
Condition Number Terminology Schematic
Zero initial entities n=0 Unoccupied
One initial entity n=1 Singly occupied
Two or more entities n>=2 Multiply occupied
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Summary of Important GMD Parameters
Mean Diameter for secretion and Growth assays from 15-70 µm.
Size determined by:
- Microdrop maker rotation speed,
- Viscosity of emulsion matrix,
- Amount of surfactant.
Number of cells per GMD approximated by Poisson statistics.
High permeability.
GMDs handle just like cells.
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Please look at the Publications Page for a list of references about GMDs.