Protocol | Published:

Three-dimensional cell culturing by magnetic levitation

Nature Protocols volume 8, pages 19401949 (2013) | Download Citation

Abstract

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

Introduction

In living tissue, cells exist in 3D microenvironments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells1,2,3,4,5. Although standard 2D or monolayer cell culture has been crucial for the development of modern biology, it inadequately recreates the natural environment within which cells reside. This lack of fidelity to the native tissue can be a severe limitation in many situations, including drug screening for toxicity and efficacy6,7,8,9. This limitation has led to the development of in vitro 3D cell culture techniques designed to provide a more physiologically relevant cellular environment that could potentially improve basic research and the drug discovery process1,2,3,4,5,10,11,12.

We recently developed the MLM to produce 3D cell cultures13. In this method, a magnetic nanoparticle assembly comprising gold nanoparticles, iron oxide and cell-adhesive peptide sequences is delivered to cells in 2D culture to render these cells magnetic13,14,15,16. When unattached and suspended in liquid, the cells can be manipulated with the external application of magnetic forces13. In particular, cells in a Petri dish or in a multiwell plate can be levitated to the air-liquid interface13. At the air-liquid interface, the cells interact and aggregate together into larger structures while synthesizing ECM proteins like collagen, fibronectin and laminin17. Overall, the MLM can be used to create 3D cell cultures with physiologically relevant ECM. Furthermore, the particular components of this method are nontoxic, do not affect proliferation and do not induce an inflammatory response by the cultured cells13,17. The protocol describes the creation of 3D cultures using the MLM, as well as other common techniques adapted to the use of magnetically levitated 3D cultures, including medium replacement, imaging, handling and IHC. We also show typical results seen when applying the MLM to various cell types.

Applications of the method

The MLM generally uses the magnetic nanoparticle assembly to promote delivery of the magnetic nanoparticles, making the MLM broadly applicable to most cell types13,17,18. Indeed, the MLM has been successfully used to make 3D cultures with all cell types tested to date, including cell lines, stem cells and primary cells13,17,18,19,20,21,22 (Table 1). The most basic application of the MLM is to culture 3D cell cultures under different biochemical or environmental conditions, and then analyze them using common biological research techniques, such as IHC17,18 and western blotting19. The ability to magnetically manipulate 3D cultures also allows for fine spatial control and more complex environments. For example, the MLM was used to create an invasion assay between two separate cultures of human glioblastoma and normal astrocytes to investigate the mechanisms of glioblastoma invasion13,19. The MLM has also been used to create coculture models of the bronchiole17 by sequentially assembling multiple 3D cultures in a layered fashion. In addition, the MLM has been used to differentiate stem cells in 3D; 3T3-L1 preadipocytes and adipose stem cells were differentiated into adipocytes and formed into a vascularized adiposphere in coculture with endothelial cells18,21. These 3D cultures are also scalable in size, so that cultures can not only be created in 96-well plates as described in this protocol but also larger cultures can be constructed in six-well plates or Petri dishes. Overall, the MLM is a versatile tool for performing basic and complex experiments in representative 3D environments.

Table 1: Cell types that have been magnetically levitated into 3D culture.

Comparison with other methods

There are many systems for 3D cell culture, including protein gel substrates23,24,25, synthetic polymer scaffolds26,27 and spheroids28,29. However, these methods are costly, often involving extensive fabrication and time-consuming analysis. For example, some protocols for spheroids take 3–4 d for spheroid formation28,29, whereas the MLM takes about 16 h to form 3D cultures. The MLM also creates 3D cultures without an artificial protein substrate, and, in fact, can synthesize ECM during formation, as demonstrated when 3D cultures of human pulmonary fibroblasts and smooth muscle cells alone produced and extruded laminin within hours of levitation17. The MLM also does not require any specialized media or a minimum serum concentration. In general, 3D cultures are cultured using the preferred medium for 2D cultures. For example, bronchial epithelial cells have been assembled in 3D without serum, owing to their sensitivity in 2D, whereas pulmonary fibroblasts need serum to grow in 3D, just as they require it in 2D (ref. 17). Thus, the MLM is a simpler tool for creating representative 3D cell culture environments when compared with other methods.

Experimental design

In this protocol we describe how to apply the MLM to create 3D magnetically levitated cultures that can replace 2D cultures. Such cultures can be used for various applications, such as investigating the dose-dependent effects of a particular drug on a particular cell type of interest. Cells are cultured in advance to confluence in 2D, but on the day before the experiment is to start, the cells are incubated with a magnetic nanoparticle assembly overnight to allow for cell attachment to the magnetic nanoparticles (Steps 1–4). The next day, the cells are detached and resuspended in medium in a 96-well plate (Steps 5–8). A magnetic drive is placed atop the well plate to levitate the cells to the air-liquid interface, where the cells aggregate and interact to form larger 3D structures (Step 9). Once the structures are fully formed and become competent (Step 10), compounds can be added to each well at specific concentrations. At a specific time point, the 3D cultures can then be assayed similarly to 2D cultures with tests such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay17, IHC17,18 (Steps 11–23) or western blotting19. Other, more complex experiments including cocultures17,18 can be conducted as well with the MLM. In all, magnetically levitated 3D cultures can replace 2D cultures with minimal adaptation to existing protocols.

Limitations

The magnetic nanoparticles have been shown to not affect cell proliferation and metabolism or induce an inflammatory response13,17. By 8 d, cells will detach from and grow without the magnetic nanoparticles, which are retained within the culture13. Similarly, exposure to the magnetic field (30–500 G) has no major effect on cell proliferation, metabolism or inflammatory response13,17, although magnetic fields of higher strength (800–4,000 G) have been shown to influence cell behavior30,31,32. In addition, the presence of iron oxide within the magnetic nanoparticle assembly will color the 3D culture brown. Although this can enhance contrast for some imaging diagnostics, the dark color may also be viewed as a disadvantage for some applications. In particular, the use of colorimetric markers that are brown, such as IHC with 3,3-diaminobenzidine, is limited. However, common colorimetric assays, such as the MTT assay, have been successfully used on cells incubated with magnetic nanoparticles17, and fluorescence is not hindered by them either17,18. There is also possibility of cell loss due to the incomplete attachment of cells to the magnetic nanoparticles during incubation that could adhere to the bottom of the plate rather than levitate, but this limitation can be addressed with the use of ultra-low-attachment multiwell plates.

Materials

REAGENTS

Critical

  • All reagents can be purchased from other distributors and manufacturers than those listed.

  • NanoShuttle (Nano3D Biosciences, cat. no. 005-NS; or see Souza and colleagues13,15,16 for directions on how to fabricate magnetic nanoparticles yourself)

  • Cells of interest

  • Cell culture medium

    Critical

    • Use cell culture medium specific to the cell type to be turned into 3D. There is no minimum serum concentration required.

  • Trypsin-EDTA, 1× (Corning, cat. no. 25-053-CI)

  • Trypsin neutralizer solution (Invitrogen, cat. no. R-002-100)

  • Ethanol (Sigma-Aldrich, cat. no. E7023)

  • Phosphate buffered saline, pH 7.4 (PBS Sigma-Aldrich, cat. no. P5368)

  • Paraformaldehyde, 4% (wt/vol) (PFA; Electron Microscopy Sciences, cat. no. 15735-100)

  • Triton X-100 (TX-100; Sigma-Aldrich, cat. no. T8787)

  • Blocking buffer

    Critical

    • Choose a blocking serum that does not interfere with the primary and secondary antibodies used.

  • Primary antibody (use an antibody for specific antigen of interest)

  • Secondary antibody (use an antibody for the specific animal of the primary antibody)

  • DAPI (KPL, cat. no. 71-03-01)


EQUIPMENT

  • Cell culture flask, 75 cm2

  • Pipette gun

  • Pipette, 5 ml

  • Incubator (37 °C, 5% CO2)

  • Laminar flow cabinet

  • Refrigerator (4 °C)

  • Freezer (−20 °C)

  • Pipettor, 200 μl

  • Pipette tip, 20–200 μl

  • Conical tubes, 15 ml

  • Hemocytometer or cell counter

  • Magnetic drive consisting of an array of 96 neodymium magnets (Nano3D Biosciences, cat. no. 009-96WD)

  • Plastic lid insert for 965-well plates (Nano3D Biosciences, cat. no. 009-96LID)

  • Ultra-low-attachment plates, 965 well (Corning, cat. no. 3474)

  • Pasteur pipettes

  • Inverted microscope

  • MagPen three-pack (comes with 0.125-inch inner diameter (i.d.) × 0.200-inch outer diameter (o.d.) Teflon pen, 0.125-inch o.d. rod magnet; Nano3D Biosciences, cat. no. 010-MPT)

  • Aluminum foil

  • Coverslips


REAGENT SETUP

NanoShuttle

  • NanoShuttle can be stored at 4 °C for 1 year.

TX-100

  • Prepare TX-100 in a 0.2% (vol/vol) solution in ultrapure H2O. The solution can be stored at 4 °C for years.

Blocking buffer

  • Prepare blocking buffer by diluting blocking serum at a 1% (vol/vol) concentration in PBS. The stock solution can be stored according to the manufacturer's directions.

Primary and secondary antibodies

  • Dilute the primary and secondary antibodies according to the manufacturer's recommended dilutions in PBS. Do not store working solutions. Freshly prepare the working solutions for every IHC staining session. Keep secondary antibody stock and working solutions away from light. The stock solutions can be stored according to the manufacturer's directions.

DAPI

  • Prepare DAPI and dilute it according to the manufacturer's recommended dilutions. Do not store the working solution. Freshly prepare the working solution for every IHC staining session. Keep the stock and working solutions away from light. The stock solution can be stored at −20 °C for 1 year.

Procedure

Incubation of cells with magnetic nanoparticles

Timing: working time 20 min, incubation time 5–16 h

Critical

  • Perform the following steps under sterile conditions using the recommended cell culture supplies for the specific cell type.

  1. Culture cells in 2D to 80% confluence using standard cell culture procedures and supplies for the specific cell type.

  2. Prepare the magnetic nanoparticle assembly by removing it from the refrigerator and thawing it at room temperature (20–25 °C) for about 15 min.

    Critical step

    • Ensure that the magnetic nanoparticles are homogenized before use, which results in an even brown color throughout the solution. If the magnetic nanoparticles are not homogenized, they must be homogenized in the vial in a sterile environment by mixing with a pipette at least ten times.

    Troubleshooting

  3. Add the magnetic nanoparticles directly to the cells and medium in the flask at a recommended concentration of 8 μl cm−2 of culture area. Gently tilt the flask back and forth to evenly distribute the nanoparticles around the flask. The medium will appear slightly darker because of the brown color of the iron oxide.

    Critical step

    • Before experimentation, optimize for cell binding to the nanoparticles by varying the volume of magnetic nanoparticles added.

  4. Put the flask back into an incubator to let the cells incubate and attach to magnetic nanoparticles for at least 5 h to overnight.

    Troubleshooting


Creating 3D cultures with MLM in 96-well plates

Timing: 25 min

  1. Aspirate the medium from the flask and detach cells by incubation with trypsin-EDTA solution for 2–5 min. Concomitantly, sterilize the well plate, magnetic drive and the lid insert with 70% (vol/vol) ethanol and bring them into the sterile environment. Once the cells are detached, add medium with serum at four times the volume of trypsin to neutralize the trypsin, and then transfer the solution into a conical tube. For more sensitive cell types, use the detachment protocol for the specific cell type. When settled or centrifuged, the cells should appear brown in color, and cell suspensions in medium should appear darker than usual (Fig. 1).

    Critical step

    • Before detachment, check the binding of cells to the magnetic nanoparticles under a microscope. Cells should appear peppered with the nanoparticles (Fig. 1).

    Troubleshooting

  2. Count the number of cells in suspension using either a hemocytometer or cell counter.

  3. Calculate the cell numbers and medium volumes needed to create 3D cultures. Typical cell numbers and medium volumes are 500–5,000 cells in 50–75 μl in 96-well plates (Fig. 2). 3D cultures can also be created in larger-well plates, such as 24-well plates. See Supplementary Figure 1 for information on how to form 3D cultures in a 24-well plate.

    Critical step

    • Optimize for the size and competence of 3D cultures by varying cell number and medium volume before experimentation.

    Critical step

    • If you are using larger plates, such as 24-well plates, optimize the cultures by varying the cell number and medium volume.

  4. Add the desired medium volume with the desired cell concentration to the wells in the plate. Gently agitate the well plate to evenly distribute the medium in the well (Fig. 3).

    Critical step

    • Use flat-bottom, ultra-low-attachment plates for maximum levitation efficiency.

    Troubleshooting

  5. Close the well plate in the following order: first, close the lid insert, then the magnetic drive and finally, the well-plate lid. Move the well plate to the incubator. 3D structures will begin to form within 15 min–1 h. These cultures should appear dense and brown, and should levitate at or slightly below the air-liquid interface (Fig. 4).

    Troubleshooting

Figure 1: Human pulmonary fibroblasts before and after incubation with magnetic nanoparticles.
Figure 1

The cells maintained their morphology but became peppered with nanoparticles after incubation. Scale bar, 100 μm.

Figure 2: Magnetically levitated 3D cultures of A549 cells.
Figure 2

Shown are different cell numbers (1,000–50,000 cells) and medium volumes (300 or 400 μl) after 0, 4 and 7 d. Scale bar, 500 μm.

Figure 3: Magnetic levitation in 96-well plates.
Figure 3

(a,b) First, take a 96-well plate (a) and add 50–75 μl of medium with cells to each well (b). (ch) Next, cover the plate with a 96-well white lid insert (c,d), 96-well magnetic drive (e,f) and lid (g,h). (i) The plate lid can then be annotated and the plate can be transferred into an incubator.

Figure 4: Magnetically levitated 3D cultures levitating in medium in a 24-well plate.
Figure 4

Note the brown color of the cultures.


Culturing 3D magnetically levitated cultures

  1. Maintain the 3D cultures in an incubator (37 °C, 5% CO2) for the length of the experiment. Medium should be replaced (option A) at regular intervals depending on the protocols for the specific cell type and experiment. 3D culture growth should be monitored by imaging regularly (option B). These cultures can also be transferred between plates (option C) for imaging or staining. When you wish to fix the cells and perform IHC, proceed to Step 11.

    1. Replacing medium in well plates with 3D cultures

      1. Sterilize the outside of the well plate with 70% (vol/vol) ethanol and bring it into a sterile environment. Open the well plate, and move the lid insert, magnetic drive and lid away from the plate, with each component turned upward.

      2. Take the magnetic drive and move it underneath the well plate with the magnets facing upward. The 3D cultures should be attracted by the magnet and move to the bottom of the well plate. Position the plate such that the magnets are off-center within the wells and that there is sufficient space to remove medium without damaging the 3D culture (Fig. 5).

        Critical step

        • Ensure that the cultures are at the bottom of the wells.

        Troubleshooting

      3. Aspirate the medium out of the wells and gently replace the medium.

        Troubleshooting

      4. Remove the magnetic drive from underneath the plate, and then cover the plate in the order described in Step 9. Move the plate back into the incubator.

      Timing: 5 min

    2. Imaging 3D cultures in a well plate with an inverted microscope

      1. Follow Step 10A(i) to open the plate in a sterile environment (Supplementary Fig. 2).

      2. Replace the lid insert and lid atop the well plate. Remove the plate from the sterile environment and move it to a microscope stage.

        Critical step

        • The lid insert is translucent, so cultures can be viewed without the need to remove the lid insert from the plate. If the culture is difficult to image with the lid insert on, remove the lid insert in a sterile environment and follow Step 11.

        Troubleshooting

      3. Return the plate to the sterile environment. Repeat Step 9 to close the plate and move the plate back into the incubator.

      Timing: 5 min

    3. Handling and transferring 3D structures with a Teflon pen

      1. Sterilize the Teflon pen and rod magnet with 70% (vol/vol) ethanol and move them into a sterile environment.

      2. Assemble the Teflon pen by inserting the rod magnet. The pen can be handled with gloved hands or plastic forceps.

        Troubleshooting

      3. Repeat Step 10A(i) to open the plate of 3D cultures in a sterile environment.

      4. With the Teflon pen facing downward, reach into the well to pick up the 3D culture. The 3D culture should be attracted and attached to the Teflon pen. Lift the pen from the well plate and remove the rod magnet from the pen. The 3D culture should stay attached to the Teflon pen (Fig. 6).

        Troubleshooting

      5. With the magnets facing upward, place the magnetic drive underneath the new well plate to which the 3D culture is being transferred.

      6. Lower the pen with the 3D culture still attached to the bottom of the new well. The magnetic drive should attract the 3D culture off the pen to bottom of the well (Fig. 6).

        Troubleshooting

      7. Gently add medium to the well.

      8. Repeat Step 10A(iv) to close the plate and move the plate into the incubator.

      Timing: 5 min

Figure 5: Replacing medium with magnetically levitated 3D cultures.
Figure 5

(a) Place the 96-well plate on a magnetic drive, but leave it off-center so that cells are held on one side of the well. (b) Medium can then be replaced in the well away from the culture without damaging it.

Figure 6: Transferring 3D cultures from a 24-well plate to a 96-well plate using the Teflon pen.
Figure 6

(a,b) Assemble the pen (a) and use it to pick up the 3D culture from the 24-well plate (b). (c,d) With the culture attached to the pen (c), remove the magnet from the pen (d). (e) The 3D culture should still stay on the pen. (f,g) Move to the 96-well plate with a 96-well magnetic drive underneath it (f) and place the pen in a well (g). (h,i) The magnet underneath the plate (h) should pull the 3D culture down to the bottom of the well plate (i).


Fixing 3D cultures for IHC

Timing: working time 5 min, fixation time 15 min–4 h

  1. Follow Step 10A(i–iii) to replace the medium with PBS. Aspirate and then wash with PBS once more.

  2. Remove the magnetic drive from underneath the plate.

  3. Add your preferred fixative, such as 4% (wt/vol) PFA, and fix the cultures for 15 min–4 h at room temperature depending on the size of the culture (small cultures will require less fixation time).

    Critical step

    • This and the following steps can be performed in a nonsterile environment.

  4. After fixation, repeat Step 11 to remove the fixative and wash the 3D culture with PBS twice.

    Pause point

    • If you wish to store the fixed cultures to use later, add PBS to each well and close the plate with the plate lid. Wrap the plate in paraffin film and store it at 4 °C for later use. Fixed cells can be stored at 4 °C for several months.


Whole-mount IHC of 3D cultures with fluorescence

Timing: working time 45 min, incubation time 4–16 h

  1. Follow Step 10C to transfer the fixed 3D culture to a new 96-well plate. Leave the magnetic drive underneath the plate for now.

    Critical step

    • Although this protocol describes the IHC staining of whole-mounted 3D cultures, these cultures should be treated as a tissue that can also be frozen or paraffin-embedded for sectioning using routine protocols17. Follow standard protocols for processing, embedding, sectioning and staining tissue.

  2. For intracellular antigens, permeabilize the cell membrane with 0.2% (vol/vol) TX-100 for 15 min. Thereafter, repeat Step 11 to remove the TX-100 solution and wash the culture with PBS five times. For this and the following steps, the 3D cultures can be stained in the same well for the remainder of the experiment either with the magnetic drive underneath the plate to maintain culture structure, or without the magnetic drive to allow for solutions to penetrate underneath cultures. The 3D cultures can also be transferred to a new well after incubation with each solution (follow Step 10C).

    Troubleshooting

  3. Add blocking buffer to the culture and incubate it for 1 h at room temperature. Blocking will prevent nonspecific antibody binding. Bring the plate back onto the magnetic drive, and for experimental wells, remove the blocking buffer.

  4. For experimental wells, remove the blocking buffer by following Step 11. Add the primary antibody solution to incubate it for either 1 h at 37 °C or overnight at 4 °C. Incubate the negative control wells with the blocking buffer.

    Critical step

    • Perform IHC using the manufacturer's recommended dilutions for the specific antigen of interest.

  5. In all wells, repeat Step 11 to aspirate all solutions and wash the cultures twice with PBS. Remove the magnetic drive and add the fluorescently labeled secondary antibody solution to incubate for 1 h at room temperature.

    Critical step

    • Check that the target animal of the secondary antibody corresponds with that of the primary antibody and does not match the animal source of the blocking buffer.

  6. Repeat Step 11 to aspirate the secondary antibody solution from the wells, and wash the cultures twice with PBS. Add DAPI to counterstain the nuclei, and then incubate the cells for 15 min at room temperature.

  7. Repeat Step 11 to aspirate the DAPI solution and wash the cultures twice with PBS. Leave the culture in PBS and remove the magnetic drive.

    Troubleshooting

    Pause point

    • If you wish to store the stained culture to image later, add PBS to each well and close the plate with the plate lid. Wrap the plate in paraffin film and store it at 4 °C for later use. Depending on the fluorophores used, stained cultures can be stored at 4 °C for several months. Refer to the manufacturer's instructions for storage of stained cultures.


Transferring 3D cultures from plates to coverslips for microscopy

Timing: 5 min

  1. On a magnetic drive with its magnets facing upward, place a coverslip on top of the magnets (Supplementary Fig. 3).

  2. From the well plate, pick up the sample using the Teflon pen as described in Step 10C. Deposit the 3D culture directly onto coverslip and proceed to imaging the culture.

Troubleshooting

Troubleshooting advice can be found in Table 2.

Table 2: Troubleshooting table.

Timing

Steps 1–4, incubation of cells with magnetic nanoparticles: working time 20 min, incubation time 5–16 h

Steps 5–9, creating 3D cultures with MLM in 96-well plates: 25 min

Step 10A, replacing medium in well plates with 3D cultures: 5 min

Step 10B, imaging 3D cultures in a well plate with an inverted microscope: 5 min

Step 10C, handling and transferring 3D structures with a Teflon pen: 5 min

Steps 11–14, fixing 3D cultures for IHC: working time 5 min, fixation time 15 min–4 h

Steps 15–21, whole-mount IHC of 3D cultures with fluorescence: working time 45 min, incubation time 4–16 h

Steps 22 and 23, transferring 3D cultures from plates to coverslips for microscopy with the Teflon pen: 5 min

Anticipated results

Optimization of medium volume and cell number

When creating 3D cultures with the MLM, the medium volume and starting cell number must be optimized. We created 3D cultures of A549s, which were varied according to starting cell number (1,000–50,000) and medium volume (300–400 μl) in 24-well plates (Fig. 2). With increasing starting cell numbers, the 3D structures became larger. Cultures grown in 400 μl of medium were larger than those grown in 300 μl up to 4 d, but by day 7, there was no observable difference in size between the two volumes. These results demonstrate how 3D cultures can be optimized for starting cell number and medium volume.

Growth of 3D magnetically levitated cultures over time

To illustrate the typical results that are obtained using the MLM, we magnetically levitated HepG2 cells and imaged them at time points up to 7 d (Fig. 7). Immediately after the external application of a magnetic field, cells incubated with the magnetic nanoparticles were attracted toward the air-liquid interface and aggregated. Over the next day, the cells began to synthesize ECM and form competent structures. In addition, cells proliferated without the magnetic nanoparticles, and the nanoparticles appeared to cluster as the 3D culture grew around them after 1 d of levitation (Fig. 7). In subsequent days, these cultures became mature with minimal change in morphology, shape or nanoparticle density. These results show the typical progression of growth in magnetically levitated 3D cultures.

Figure 7: Magnetically levitated 3D cultures of HepG2s.
Figure 7

(ai) After 0 min (a), 5 min (b), 15 min (c), 30 min (d), 45 min (e), 4 h (f), 24 h (g), 4 d (h) and 7 d (i). Scale bar, 250 μm.

Magnetic levitation of different cell types

When viewed under a microscope, magnetically levitated 3D cultures will vary among cell types in density of the nanoparticles, size and morphology (Fig. 8). For example, 3T3-L1 cells will tend to form small and dense clusters, whereas A549 cells will form less-dense and sparse structures. Common to 3D cultures of all cell types are the presence of the magnetic nanoparticles, as indicated by the brown color, and the dense packing of cells.

Figure 8: Micrographs of magnetically levitated 3D cultures of various cell types.
Figure 8

(a) HEK293; (b) human tracheal smooth muscle cells; (c) human pulmonary fibroblasts; (d) human glioblastoma; (e) H-4-II-E; (f) MDA-231; (g) human umbilical vein endothelial cells (HUVECs); (h) MCF-10A; (i) LNCaP; (j) HepG2; (k) A549; and (l) 3T3-L1. Scale bar, 100 μm.

IHC of magnetically levitated 3D cultures

After culture, magnetically levitated 3D cultures can be analyzed with various analytical tools, such as IHC, similarly to 2D cultures or other 3D culture systems. For example, A549 cells were levitated with 175,000 cells and 400 μl of medium in 24-well plates for 2 d before they were fixed in 4% (wt/vol) PFA. The cultures were then whole-mount stained for mucin-5AC, cytokeratin-19 and E-cadherin to verify epithelial cell phenotype and function and N-cadherin to view cell-cell interactions. The stained cultures were imaged using a confocal microscope. These cultures stained positively for all markers, indicating the maintenance of epithelial phenotype and function when cultured in 3D and demonstrating that the MLM maintains phenotype overall (Fig. 9). These results also demonstrate that immunofluorescence staining is possible in these cultures with no apparent interference from the magnetic nanoparticles.

Figure 9: Immunohistochemical staining patterns of 3D cultures of A549s for mucin-5AC (Abcam, cat. no. ab3649, 1:100 dilution), cytokeratin-19, (Abcam, cat. no. ab15463, 1:100 dilution), E-cadherin (Invitrogen, cat. no. 13-1700, 1:200 dilution), and N-cadherin (Invitrogen, cat. no. 33-3900, 1:100 dilution) after 2 d of culture.
Figure 9

These 3D cultures were constructed with 175,000 cells per culture in 400 μl of medium. Positive staining patterns for mucin-5AC, cytokeratin-19 and E-cadherin verified epithelial phenotype and function, whereas N-cadherin demonstrated cell-cell interactions within the 3D culture. Scale bar, 100 μm.

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Acknowledgements

This work was supported by a US National Science Foundation (NSF) Small Business Innovation Research Award Phase I (0945954) and Phase II (1127551) from the NSF IIP Division of Industrial Innovation and Partnerships, and by an award from the State of Texas Emerging Technology Fund.

Author information

Author notes

    • William L Haisler
    •  & David M Timm

    These authors contributed equally to this work.

Affiliations

  1. Department of Bioengineering, Rice University, Houston, Texas, USA.

    • William L Haisler
  2. Department of Physics, Rice University, Houston, Texas, USA.

    • David M Timm
    •  & T C Killian
  3. Nano3D Biosciences, Houston, Texas, USA.

    • Jacob A Gage
    • , Hubert Tseng
    •  & Glauco R Souza

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Contributions

W.L.H. and D.M.T. contributed equally to this protocol by running the majority of experiments, gathering the bulk of the data presented in this protocol and preparing the manuscript. J.A.G. both helped in gathering data and images for this protocol. H.T. helped to write the manuscript. T.C.K. and G.R.S. invented and optimized the technique described in this protocol.

Competing interests

The University of Texas M. D. Anderson Cancer Center (UTMDACC) and Rice University, along with their researchers, have filed patents on the technology and intellectual property reported here. If licensing or commercialization occurs, the researchers are entitled to standard royalties. G.R.S. and T.C.K. have equity in Nano3D Biosciences, Inc. UTMDACC and Rice University manage the terms of these arrangements in accordance with their established institutional conflict-of-interest policies.

Corresponding author

Correspondence to Glauco R Souza.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Magnetic levitation in 24-well plates. First, take a 24-well plate (A) and add 300-400 μL of media with cells to each well (B). Next, cover the plate with a 24-well white lid insert (C,D), 24-well magnetic drive (E,F), and lid (G,H). The plate lid can then be annotated and the plate can be transferred into an incubator (I).

  2. 2.

    Supplementary Figure 2

    Imaging magnetically levitated 3D cultures in a 96-well plate. First, bring the plate into a sterile environment (A), and remove the lid (B), then magnet (lid insert if necessary) (C). Next, replace the lid atop the 96-well plate (D) and move the plate out of the sterile environment (E) onto a microscope stage to image (F).

  3. 3.

    Supplementary Figure 3

    Transferring magnetically levitated 3D cultures from a well plate to a coverslip for imaging. Take a coverslip and place atop the 96-well magnetic drive with the magnets facing upwards (A,B). Next, pick up a 3D culture from a well plate with a Teflon pen (C). With the culture attached to the pen (D), remove the magnet from the pen (E). The 3D culture should still stay on the pen (F). Finally, place the culture on the coverslip by moving it close to the magnetic drive over the coverslip (G).

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DOI

https://doi.org/10.1038/nprot.2013.125

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