Question about spheroid assay

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swdbell's picture
Question about spheroid assay

I have some question about angiogenesis method. I have a problem in experiment.
I do Spheroid assay for 3D spheroid by Hanging drop technique. Using matrix by collagen from rat tails so my matrix is not clear   I think it participate of collagen,   because it has a white fibrous whan see in well and debris in microscope.
From this problem impact for spheroid is not good,not circle,       cell of this expose form there in next time so don't see abnormal shape in first day.         I think this problem happens form collagen hasn't well of polymerization.
How can do it ?       I do collagen on ice 3-5 minitue.

Thanks you


Cecilia Suarez
Cecilia Suarez's picture
Hello swdbell

Hello swdbell

I'm enclosing here a protocol about growth of spheroids in collagen I gels. You will need to make the necessary changes because you have other type of collagen, but you can get a start point here. I couldn't attach the whole paper because it is too heavy, but it is: Kaufman LJ et al, Biophysical Journal 89: 635-650, 2005. Good luck!

Collagen matrix preparation
The collagen matrices are prepared from the following ingredients: a stock
solution at 2.9 mg/ml collagen I (Vitrogen, Cohesion Tech, Palo Alto, CA),
MEM 103 solution (Gibco Invitrogen) and/or DMEM 1X solution (Gibco),
10% w/v sodium bicarbonate, fetal bovine serum (JRH Biosciences),
penicillin/streptomycin (Gibco Invitrogen), and NaOH (1 N). Enough
collagen is used to attain the desired final concentration (0.5–2.5 mg/ml),
and 10% MEM, 100 U/ml penicillin, and 100 mg/ml streptomycin are added.
NaOH is added to bring the pH to 7.4, 2 mg/ml Na(CO3)2 is added to buffer
the gel, and the solution is topped off with deionized water to bring the total
volume to 2.5 ml. The solution is well mixed and kept at 4C, degassed for
;30 min, and placed in one of three types of sample cells: 1-cm3 plexiglass
cubes, 1-cm diameter plexiglass cylinders of height up to 1 cm, or shorter
(;2-mm) glass chambers fully sealed with UV epoxy. In all cases, a thin
glass coverslip forms the bottom of the sample cell. No differences were
found in the structure of the collagen gels in the short versus the tall glass
chambers, and the bare collagen experiments (experiments with no
implanted cells) were done in the thin sample cells. Experiments on GBM
in collagen matrices were done in the thick cubic or cylindrical cells in both
anchored and relaxed gels. To prepare the sample cells to hold anchored
gels, in which the collagen does not pull far from the walls as the solution
gels and in which global remodeling by the cells is minimized, they are lined
with nylon mesh to which the collagen anchors. For experiments in relaxed
gels, no nylon mesh is used.
In the GBM/collagen samples, 400 mL of collagen solution is added to
the chambers. One to three spheroids are placed in each sample cell, and the
sample cells are covered and incubated at 37C and 5% CO2. This begins
polymerization of the collagen while maintaining the health of the cells. The
MTSs generally sediment to the lowest 100–200 mm of the sample cell,
within the working distance of a typical high-numerical aperture objective.
Full gelation occurs within 1 h, and a superlayer of culture medium is then
added to maintain moisture and pH. The superlayer is changed at least every
48 h. In most cases, the spheroids remain healthy for 4–6 days. During
brightfield and phase-contrast microscopy, the sample cells are in a
temperature- and CO2-controlled chamber. During confocal and CARS
microscopy, the sample cells are on a temperature-controlled, but not CO2-
controlled, stage. Coverslips are affixed to the sample cells with mineral oil
to prevent air exchange and help maintain pH during these measurements.
Because the longest the samples were kept on the microscope stand during
these types of microscopy was 3 h at a time, deleterious effects due to pH
changes were not observed.

swdbell's picture
Thank you about your answer.

Thank you about your answer. I think because your protocol have NaHCO3 . But in my protocol don't have it. Are you agree with me ? In collagrn gel ingredient

-collagen stock solution
-Cell mix with media

Sometime NaHCO3 will helping mixiing solution. I use 24-well plate for do not using plexiglass. How about your opinion ? All of this set on ice, wait collagen polymerization 30 minute . Do you think shortly time or ok?

Thanks you


Cecilia Suarez
Cecilia Suarez's picture
What exactly are your

What exactly are your problems? Your spheroids are not round and collagen doesn´t polimerize? They are two separate problems because you have to make the spheroids before you put them on collagen. Or perhaps spheroids loss their shape when placed on collagen? Have in mind that not all cell lines make well-rounded spheroids. Does your cell line make rounded spheroids by the hanging drop technique? If you have it solved, then, separatelly, test your collagen polimerization protocol (without spheroids and testing salt compent). Once you have both parts solved, then test mixing them without damaging the spheroids. I don´t think that a 24-well-plate would be a problem. For the rest, follow your most reliable protocol. Good luck!

mcuddihy's picture
 I'm not sure if this will be

 I'm not sure if this will be helpful to your overall experiment goals, but you can try to not use a matrix at all. There are hanging drop plates that are designed to form uniform spheroids. They are described in Tung YC et al. Analyst 136(3): 473-478, 2011. Link here.

I'm not sure they've been used for angiogenesis assays, though. I hope this is helpful; good luck!