supplementary materials three-dimensional tissue culture ... · 2 nature nanotechnology |...

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nnano.2010.23

nature nanotechnology | www.nature.com/naturenanotechnology 1

Supplementary Materials

Three-dimensional Tissue Culture Based on Magnetic Cell

Levitation

Glauco R. Souza1,9, Jennifer R. Molina2, Robert M. Raphael3, Michael G. Ozawa1,

Daniel J. Stark4, Carly S. Levin5, Lawrence F. Bronk1, Jeyarama S. Ananta6,

Jami Mandelin1, Maria-Magdalena Georgescu2, James A. Bankson7, Juri G. Gelovani8,

T. C. Killian4,*, Wadih Arap1,*, and Renata Pasqualini1,*

1 David H. Koch Center, The University of Texas M. D. Anderson Cancer Center,

Houston, Texas 77030 2 Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer

Center, Houston, Texas 77030 3 Department of Bioengineering, Rice University, Houston, Texas 77005 4 Department of Physics and Astronomy, Rice University, Houston, Texas 77005 5 Nano3D Biosciences, Inc., Houston, TX 77030 6 Department of Chemistry, Rice University, Houston, Texas 77005 7 Department of Imaging Physics, The University of Texas M. D. Anderson Cancer

Center, Houston, Texas 77030 8 Department of Experimental Diagnostic Imaging, The University of Texas M. D.

Anderson Cancer Center, Houston, Texas 77030 9 Present address: Nano3D Biosciences, Inc., Houston, TX 77030

*e-mail: [email protected]; [email protected]; [email protected]

© 2010 Macmillan Publishers Limited. All rights reserved.

2 nature nanotechnology | www.nature.com/naturenanotechnology

SUPPLEMENTARY INFORMATION doi: 10.1038/nnano.2010.23

Nanoparticle description and synthesis

Gold nanoparticle solutions (50 ± 8 nm diameter) were prepared following the common

citrate-reduction1 procedure (molar ratio of 0.8:1.0 of sodium citrate:Au(III) chloride;

Sigma-Aldrich). Magnetic iron oxide (MIO) nanoparticles (Fe3O4, magnetite) are

polydisperse and generally within the ~10-100 nm size range. Notably, whereas

magnetite nanoparticles <30 nm are superparamagnetic, our preparation displays remnant

magnetization characteristic of bulk ferrimagnetism2.

Au-phage-MIO hydrogel characterization

Prior to experimental use, each supernatant was shown to be nanoparticle-free as

evidenced by light extinction measurements in the visible region, data indicative that all

metal nanoparticles were incorporated into the resulting hydrogel.

To evaluate the magnetic (MIO-containing) hydrogel, we first used elastic light

scattering. Darkfield microscopy studies (Supplementary Fig. S1) revealed that the basic

microstructure was similar to that of the previously characterized MIO-free hydrogel3.

The strong red-color scattering is characteristic of Au nanoparticles. The color and

microstructure resemble the MIO-free Au-phage hydrogel assembly3,4. Also, MIO-

containing hydrogels appear to be predominantly stabilized by electrostatic interactions

(Fig. 1b, c), a result consistent with the established biochemical behavior of MIO-free

hydrogels3. Indeed, gold and magnetite are attracted to positively charged phage particles

because both nanoparticles acquire negative charge under aqueous solution (pH 6.0)

conditions4,5. However, we did observe that admixtures of MIO and phage are unable to

self-assemble into gold-free hydrogels (data not shown), a result indicative that MIO

nanoparticles are less effective at cross-linking per se.

2




Because MIO nanoparticles are a commonly used MRI contrast enhancer6, we

subsequently showed their presence within the hydrogel through T2*-weighted MRI (Fig.

1d). We also compared iron oxide content of supernatants from solutions of MIO-

containing hydrogels or pure (negative control) iron oxide through Inductively-coupled

Plasma Atomic Emission Spectroscopy (ICP-AES) as another confirmatory methodology

(Supplementary Fig. S2). Au-phage-MIO hydrogel was prepared (see methods), resulting

in a MIO concentration of 0.15 mg/ml. A solution of free MIO in water (0.15 mg/ml) was

also prepared (Supplementary Fig. S2, suspended). The samples were allowed to settle

ON (Supplementary Fig. S2, settled), and the hydrogel fragments settled much more than

the free MIO nanoparticles in solution. A clear supernatant in the hydrogel indicates the

extent to which MIO nanoparticles are incorporated. To quantify this, we analyzed

supernatants of each sample with ICP-AES. The concentration of MIO remaining in the

hydrogel supernatant was only 6% of the concentration in the supernatant of the free

MIO. For ICP-AES sample preparation, a known volume of each sample was digested

with chloric acid (~ 25 %) [note: not hydrochloric acid] on a hot plate until a solid

residue was formed. The solid residue was dissolved in 2% HNO3 and analyzed by ICP-

AES to determine iron concentration. Yttrium was used as an internal standard. In

addition to the samples, a control sample was prepared in the same way but with none of

the test solution. Analysis was performed with a Perkin-Elmer Optima 4300 DV ICP-

AES. ICP-AES on 4x105 cells immediately after levitation was performed as described

above to determine the average amount of magnetite present within each cell. We

observed approximately 70 pg/cell.

3




Supplementary Fig. S1. Elastic light scattering (dark field microscopy) image of Au-phage-MIO hydrogel (constant light intensity). Scale bar, 2 µm.

Supplementary Fig. S2. Differential settling of Au-phage-MIO hydrogel versus free MIO nanoparticles. Both solutions start with an equal MIO concentration (0.15 mg/ml). After an equal time, the supernatant for the hydrogel shows essentially no MIO because it has presumably been incorporated into the hydrogel.

4




Theoretical gravitational force estimates for 3D culture systems

order to levitate a cell, the magnetic force on the cell (Fmag) must exceed the force of

gravity (Fgrav) minus the buoyancy force due to the media displaced by the cell (Fbuoy),

Fmag> Fgrav- Fbuoy. We estimate the forces as Fbuoy= ρwaterVg = 21 pN, where ρwater = 1

g/cm3 is the density of water, V = 2.1 x 10-9 cm3 is the volume of a cell with approximate

diameter of 16 µm, and g = 9.8 m/s2 is the gravitational acceleration. The gravitational

force is Fgrav = ρcellVg = 23 pN, for a cell density taken as 1.08 g/cm3. The cells are

almost neutrally buoyant and the difference force is Fgrav- Fbuoy = 2 pN.

The magnetic force on the symmetry axis of the magnet is given by Fmag =

m∇B,where ∇B is the magnitude of the gradient of the magnetic field and m is the total

magnetic moment due to all the magnetite in the cell (70 pg/cell). We recorded the

magnetization curve (Supplementary Fig. S3) of the magnetic nanoparticles at 300 K in a

Quantum Design Magnetic Properties Measurement System (QD MPMS; T =1.8-400 K,

Hmax = 5.0 T).

Supplementary Fig. S3: Magnetization as a function of magnetic field at constant temperature (300 K) for the magnetic iron oxide (MIO) nanoparticles. Inset shows the magnetic hysteresis. Note: 1 T=10,000 G

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0-100-80-60-40-20

020406080

100

In

-0.2 -0.1 0.0 0.1 0.2-80

-40

0

40

80

M (e

mu/

g)

H (T)

M (e

mu/

g)

H (T)

300 K

5




The magnetic field for the weakest magnet (Fig. 4a) is approximately 200 G at the

e conditions

ansfected into

Bosc ce post-

bottom of the petri dish, and the gradient is approximately 50 G/mm. Thes

yield a magnetization of about 20 emu/g and a force of 7 pN, which is just enough to

levitate the cells off the bottom of the dish. This setting is the lower limit of magnetic

force used in the experiment.

Plasmids and cell transfection

The mCherry and eGFP in the retroviral vectors pCXb and pCXp were tr

lls to produce retrovirus containing supernatants which were collected 48 h

transfection and used for infection of normal astrocytes, neural stem cells, or

glioblastoma cells, respectively7. The cells were treated and maintained in selection

media 48 h after infection and express the fluorescent protein in a stable manner.

6




3D cell

t

ated under standard tissue culture conditions (Fig. 2a). In

revious studies, we used confocal microscopy to validate that targeted phage particles

nd gold nanoparticles adhere to outer mammalian cell membranes and undergo receptor-

n overnight incubation, the same phenomenon was

d upon the

re

d size)

by the average volume of a single cell (~1.0 nL).

3D cell levitation preparation without cell surface attachment

A procedure for 3D culture through magnetic levitation without surface attachment is

also feasible. Suspended cells can be incubated with Au-phage-MIO for 15 min, after

which they are cultured in 3D by magnetic levitation (Supplementary Fig. S4a). The yield

of levitated vs. non-levitated cells is influenced by the amount of Au-phage-MIO,

incubation time, strength and gradient of magnetic field, and distance from magnet to

bottom surface. We show mCherry-transfected normal human astrocytes cultured for 15

min (Fig. S4b) and 48 h (Fig. S4c).

levitation preparation with cell surface attachment

The standard procedure for magnetic cell levitation starts with cell attachment to the fla

bottom surface of a culture plate, and MIO-containing targeted hydrogel was dispersed

through pipetting. Size distribution of hydrogel fragments was not found to be critical.

The admixture was then incub

p

a

mediated internalization3. After a

observed (data not shown); neural stem cells were subsequently rinsed with PBS and any

hydrogel remnants were removed (Fig. 2b). The neural stem cells were detached from

the surface with a standard trypsin:EDTA treatment3,4 and a magnet was place

tissue culture dish (Fig. 2c). The number of cells in the levitated multicellular structu

was estimated by dividing the estimated volume of the structure (from its shape an

7




8




Location of Au and MIO nanoparticles and necrosis in cellular assembly

While it is known that mammalian cells eventually process biological material such as

phage8 , the cellular fate of metal nanoparticles, such as MIO, is far less established9.

Transmission electron microscope (TEM) studies showed detectable intracellular

nanoparticles after 24 h, but after approximately one week in 3D culture, magnetically

levitated human glioblastoma cells predominantly release nanoparticles into the media

and/or extracellular matrix (Supplementary Fig. S5). In addition, viable multicellular

assemblies dropped when the magnet field was removed but re-levitated when the magnet

field was reapplied after long-term in culture. While the molecular mechanism(s) for

these observations (such as secretion, apoptotic cell death, or a combination) remains to

be determined, the apparent “entrainment” of metal nanoparticles in the assembly may

partially explain the ability to levitate 3D cultures for relatively extended periods of time.

TEM of cross-sections of spheroids of human glioblastoma cells grown through

magnetic levitation show the location of nanoparticles at different stages (Supplementary

Figs. S5-S6). After 24 h of levitation, the bulk of nanoparticles are contained in the cell

cytoplasm, consistently with previous reports3,10. After 8 days in culture, cells have

processed the nanoparticles and they appear in the extracellular matrix (ECM). Cellular

division and growth of the spheroid presumably leads to a differential distribution of the

nanoparticles (preferentially present in the center of the spheroid rather than in the outer

region). Multicellular structures of human glioblastoma cells were fixed in 10 mM PBS

containing 1% glutaraldehyde after 24 h and 8 d of magnetic levitation. These structures

were then placed on a nickel mesh grids previously coated with Formvar and evaporated

with carbon were floated on drops of 0.1% poly-L-lysine (Sigma Diagnostics) on

9




parafilm for 5 min. Excess solution was removed from the grid by carefully touching the

f

as

edge of the grid onto filter paper. The grids were not allowed to dry completely in any o

the steps. The grids were floated on drops of sample on parafilm for 1 h. Excess fluid w

removed and the grids then were floated on drops of 0.02% BSA containing 1%

ammonium molybdate in distilled water (pH 7.0) for 1 min. Excess fluid was removed,

and the grids were allowed to dry ON. Images were captured by a transmission electron

microscope (JEM-1010, JEOL) fitted with an AMT Advantage (Deben UK Limited,

Suffolk, U.K.) digital charge-coupled device camera system.

Supplementary Fig. S5. TEM of human glioblastoma cells grown under magnetic levitation. (Left) After 24 h of culture, nanoparticles (black) are found inside the cells. (Middle) After 8 d of culture, nanoparticles remain in the central region of the tissue spheroid but largely in the ECM. (Right) The outer regions of the spheroid (8days of culture) contains far fewer detectable nanoparticles. Scale bar, 5 µm.

10




Su d after 8 axially sy panel) surrounded by viable cells (right panel). No necros 24 hrs of growth, as shown in Supplementary Fig. S5. Scale bar, 10 µm.

pplementary Fig. S6. TEM analysis of a typical 3D hydrogel-grown tumor spheroidays of growth. Malignant glioma cells grown under magnetic levitation create an mmetric structure (left panel), with central necrosis (middle

is was observed in culture after

11




12

Scanning electron microscopy

Multicellular structures of human glioblastoma cells were fixed, critical-point dried, and

coated with Au/Pd11. SEM images (Supplementary Fig. S7) were captured with the JSM

5900 scanning electron microscope (JEOL USA, Inc., Peabody, MA) equipped with

backscatter electron detector and digital camera. Images show the three-dimensional

nature of the tissue cultures.

Supplementary Fig. S7. SEM of human glioblastoma cells grown under magnetic levitation for 24 h (Left) and 8 d (Right). Scale bar, 100 µm.




Long-term levitated culture

3D levitated cultures of GFP-expressing human glioblastoma cells maintained for 2 days

demonstrate robust signal intensity (Supplementary Fig. S8a). Twelve weeks later, large

cell masses (Supplementary Fig. S8b) retain strong GFP fluorescence signal indicative of

viable cells within the spheroid.

Supplementary Fig. S8. Fluorescence photomicrographs of levitated GFP-expressing human glioblastoma cultured for (A) 2 days and (B) 12 weeks. After 12 weeks cells show strong GFP fluorescence signal indicating cell viability. Scale bar, 200 µm.

13




Effect of 3D cell culture on N-cadherin expression

Supplementary Fig. S9. Immunofluorescence detection of N-cadherin (red, fluorescence from Alexafluor 555) and nuclei staining (blue, fluorescence from DAPI) staining of human glioblastoma cell culture attached to a glass slide cover slip (2D cultured for 48 h). The conditions are no Au-phage-MIO and no magnetic field (upper left), Au-phage-MIO treatment but no magnetic field (lower left), no Au-phage-MIO but with magnetic field (upper right), and Au-phage-MIO treatment and magnetic field but with the magnet below the culture plate so the cells grow on the bottom surface in 2D. None of these control conditions show the enhanced N-cadherin expression present in levitated 3D culture or mouse xenograft shown (see also Fig. 4). In these experiments, the magnitude of the magnetic field was equivalent to the one experienced by levitated cells.

We observed no detectable alteration in the N-cadherin expression (Supplementary Fig.

S9) in attached human glioblastoma cells due to any combination of Au-phage-MIO

treatment and magnetic field that did not result in 3D levitated culture.

14



supplementary informationdoi: 10.1038/nnano.2010.23

Calculation of magnetic fields

The magnetic field of a ring magnet was calculated by numerically integrating the Biot-

Savart law using Mathematica (Wolfram Research Inc.; Champaign, IL), treating the

magnet as a ring of constant magnetization M

parallel to the symmetry axis. This led to

a magnetic field at a position r of '

')'()'()( da

rrrrrKrB

S∫ −

−×= 3

0

4

πµ

. The integration

extended over the surface of the magnet, S , and the bound surface charge is nMK ˆ×=

,

where n̂ is the unit vector pointing out of the magnet and normal to the surface

everywhere. The magnitude of M

was adjusted so that the calculation matched field

measurements made with a Hall probe.




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