3D culture by magnetic levitation

Biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes 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 cells. 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 efficacy. 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 process.

MLM is a recently developed method to produce 3D cell cultures. 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 magnetic. When unattached and suspended in liquid, the cells can be manipu­lated with the external application of magnetic forces. In particu­lar, cells in a Petri dish or in a multiwell plate can be levitated to the air-liquid interface. At the air-liquid interface, the cells interact and aggregate together into larger structures while synthesizing ECM proteins like collagen, fibronectin, and laminin. 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 cells. 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.

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 a 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) assay, IHC (Steps 11–23) or western blot­ting. Other, more complex experiments including cocultures can be conducted as well with the MLM. In all, magnetically levi­tated 3D cultures can replace 2D cultures with minimal adapta­tion to existing protocols.

Materials

Reagents

  • NanoShuttle
  • Cells of interest
  • Cell culture medium: Use cell culture medium specific to the cell type to be turned into 3D. There is no minimum serum concentration required.
  • Trypsin-EDTA
  • Trypsin neutralizer solution
  • Ethanol
  • Phosphate buffered saline, pH ~7.4
  • Paraformaldehyde, 4% (wt/vol)
  • Triton X-100
  • Blocking buffer: 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: 2-(4-amidinophenyl)-1H -indole-6-carboxamidine

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
  • Plastic lid insert for 965-well plates
  • Ultra-low-attachment plates, 965 well
  • Pasteur pipettes
  • 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 )
  • Aluminum foil
  • Coverslips

Reagent Setup

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

CAUTION : Do not freeze it.

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. 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.
  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.

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

CAUTION : Longer incubation times will result in the nanoparticles detaching from the cells.

Creating 3D cultures with MLM in 96-well plates ● Time: 25 min

  1. Aspirate the medium from the flask and detach cells by incubation with the 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 the medium should appear darker than usual.

CAUTION : Do not autoclave the magnetic drive beforehand. Magnets will demagnetize when exposed to high tempera­tures. The lid insert can be autoclaved, but take care that you keep it flat. Sterilization with 70% (vol/vol) ethanol is sufficient.

CRITICAL STEP : Before detachment, check the binding of cells to the magnetic nanoparticles under a microscope. Cells should appear peppered with the nanoparticles.

  1. Count the number of cells in suspension using either a hemocytometer or cell counter.
  2. 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. 3D cultures can also be created in larger-well plates, such as 24-well plates.

CAUTION : Too much medium in the well will bring 3D cultures closer to the magnet, which could result in cells escaping the medium rather than levitating in it.

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.

  1. 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.

CRITICAL STEP : Use flat-bottom, ultra-low-attachment plates for maximum levitation efficiency.

  1. 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.

CAUTION : Keep the plate flat during handling, as tilting the plate could bring the 3D culture closer to the magnet, where it could escape the medium and attach to the lid insert.

Culturing 3D magnetically levitated cultures

  1. Maintain the 3D cultures in an incubator (37 °C, 5% CO2) for the length of the experiment. The 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.

(A) Replacing medium in well plates with 3D cultures for 5 min

(i) 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.

(ii) 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. CRITICAL STEP Ensure that the cultures are at the bottom of the wells.

(iii) Aspirate the medium out of the wells and gently replace the medium.

(iv) 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.

(B) Imaging 3D cultures in a well plate with an inverted microscope ● TIMING 5 min

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

(ii) Replace the lid insert and lid atop the well plate. Remove the plate from the sterile environment and move it to a microscope stage. CAUTION Keep the plate flat, as rough handling could disrupt the 3D culture.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.

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

(C) Handling and transferring 3D structures with a Teflon pen ● TIMING 5 min

(i) Sterilize the Teflon pen and rod magnet with 70% (vol/vol) ethanol and move them into a sterile environment. CAUTION Do not autoclave the rod magnet. Sterilization with 70% (vol/vol) ethanol is sufficient. The Teflon pen can be autoclaved.

(ii) Assemble the Teflon pen by inserting the rod magnet. The pen can be handled with gloved hands or plastic forceps. CAUTION Do not use forceps made of magnetic metals, which would attract the rod magnet within the pen.

(iii) Repeat Step 10A(i) to open the plate of 3D cultures in a sterile environment.CAUTION Keep the magnetic drive a good distance away from the working area to prevent it from interfering with the Teflon pen.

(iv) 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.

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

(vi) 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.

(vii) Gently add medium to the well.

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

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.

  1. 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 IHCof 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 protocols. Follow standard protocols for processing, embedding, sectioning, and staining tissue.

  1. 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).
  2. 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.
  3. 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.

  1. 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.

CAUTION From this step onward, cover the plate with aluminum foil to prevent photobleaching of the fluorescent tags.

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.

  1. 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.
  2. 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.

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

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

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 types. 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 cells. 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 IHC and western blotting. 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 invasion. 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. 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.