Our protocol offers a 3D bio-engineered model that closely affects the native tissue. This can potentially be used to discover new therapeutics and biomarkers for neuroblastoma treatment and diagnosis. The main advantage is that it creates more physiologically relevant experimental conditions to study the tumor microenvironment using a reproducible technique that achieves batch to batch consistency.
Our scaffold method can be utilized firstly, to identify novel therapeutics for neuroblastoma, and secondly, to incorporate patient-derived cells for better prediction of individual patient response. To begin, place the scaffold stored in PBS into the laminar flow hood. Use sterile tweezers to gently lift the scaffold from the corner, and press against the sidewall to remove excess PBS.
Then place the scaffold with the shiny layer facing down in the center of the well of a non-adherent 24-well plate. Label the plate with details of the cell line, seeding density, and time points. Work with the cells of one seeding density at a time, and keep the remaining cells at 37 degrees Celsius in the incubator, until ready for use.
For the cell attachment in the scaffold, use a P20 pipette with sterile tips to thoroughly mix the cell suspension, and add 20 microliters of the relevant cell suspension in the center of each scaffold, ensuring that the cell suspension remains on top of the scaffold and not on the sidewall or base of the well. Allow the cells to attach for three to five hours at 37 degrees Celsius in 5%carbon dioxide and 95%humidity. At the end of the incubation, use a P1000 pipette to slowly add one milliliter of prewarmed growth medium to each well, preventing displacement of the scaffolds, and incubate the plate overnight.
Observe the scaffolds, initially, every two to three days for color changes of the growth medium as the cells proliferate within the scaffold. Use a 10 milliliter pipette gun with slow mode to remove and discard one milliliter of the spent medium from the well. When the conditioned medium is used for the experiment, collect the spent medium of the biological replicates in a 15 milliliter centrifuge tube, and pellet down the cellular debris by centrifugation.
Transfer the supernatant to a fresh tube and store the tube at minus 80 degrees Celsius. After setting the pipette gun on the drip mode, gently add two milliliters of prewarmed growth medium to the scaffolds. Incubate the scaffold-containing plate and replenish the fresh medium for the duration of the desired growth period, as demonstrated.
In the laminar flow hood, sterilize the appropriate cell viability assay reagent by filtration through a 0.2 micrometer sterile filter into a centrifuge tube. Prewarm the sterile solution, complete growth medium, and sterile PBS in the water bath, at 37 degrees Celsius. Using sterile tweezers, transfer the scaffolds to be analyzed in a fresh 24-well plate, and label the plate with all relevant details.
Add 900 microliters of the prewarmed growth medium to each well. Then add 100 microliters of the sterile cell viability reagent to each well. For a negative control, add 900 microliters of medium and 100 microliters of the sterile cell viability reagent in a well with no scaffold.
Replace the lid on the plate, and gently rock the plate for approximately three minutes to distribute the diluted cell viability reagent throughout the well. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity. After removing the plate from the incubator, gently rock the plate for a few seconds and generate the triplicates, as demonstrated in the text manuscript.
Generate the technical triplicates by transferring 100 microliters of the incubated medium and reagent from one well of the 24-well plate into one well of the translucent 96-well plate, and perform this step a total of three times for one well. Cover the 96-well plate with aluminum foil to protect the cell viability reagent from light. Remove and discard the remaining 700 microliters of medium and reagent from the scaffolds in the 24-well plate.
Wash each scaffold twice with one milliliter of sterile PBS. Use a microplate reader to measure the absorbance at 570 and 600 nanometers, and calculate the percentage reduction of the cell viability reagents as per the manufacturer's recommendations. Statistically analyze the cell viability results, using appropriate software.
Input the biological triplicate values to produce error bars and indicate the assay variability. Perform a one-way analysis of variance test to examine the changes in cell viability over the experimental timeframe using appropriate biostatistical software. Indicate significant differences between the time points on the graphs, as described in the text manuscript.
The viability of two neuroblastoma cell lines, KellyLuc and IMR32, grown on the scaffolds, was assessed. The increased cell density resulted in more reduction of the cell viability reagent with time. The cell growth on the scaffolds was indirectly measured by quantifying the DNA extracted from the scaffolds, and the cells per sample were calculated.
The IMR32 cells showed increased growth, then KellyLuc cells, with time. The growth morphology and distribution of cells on the scaffolds were visually assessed by hematoxylin, eosin, and immunohistochemistry staining. KellyCis83 cells grew faster and infiltrated deeper into both scaffold compositions than the less invasive Kelly cell line.
IMR32 grown on nano-hydroxyapatite demonstrated a contrasting growth pattern with large, densely-packed clusters, over 14 days. The cell-specific traits were monitored by immunohistochemistry staining, followed by phalloidin and DAPI staining, and the abundance of actin was observed in Kelly and KellyCis83 cells on collagen-glycosaminoglycan scaffolds. For the in vitro cellular activity assessment, secreted levels of chromogranin A were measured, and the cells grown on collagen-glycosaminoglycan and collagen nano-hydroxyapatite scaffolds produced more chromogranin A compared to the growth on conventional 2D culture.
The chemo-resistant KellyCis83 cell line secreted more chromogranin A than the Kelly cell line. After cell attachment, cells can be treated with various therapeutics. This would provide more physiologically relevant results for cells'response to drugs than conventional 2D culture.
This technique allows researchers to better explore how cancer cells behave and react to a stimulus within the tumor microenvironment, ranging from response to therapeutics or interactions with other cell types.
