Apr 01, 2026
Seeding and culturing adherent cells on 3D broccoli-based scaffolds
- Catherine A. Sword1,
- Kristopher Kubow1
- 1James Madison University
Protocol Citation: Catherine A. Sword, Kristopher Kubow 2026. Seeding and culturing adherent cells on 3D broccoli-based scaffolds. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4p1y8lo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 13, 2026
Last Modified: April 01, 2026
Protocol Integer ID: 313217
Keywords: 3D, cell culture, plant-based, decellularized, broccoli, cell biology, bioengineering, fibronectin, scaffold, adherent cells on 3d broccoli, culture adherent cells on scaffold, decellularized broccoli stalk tissue, broccoli stalk tissue, culturing adherent cell, culture adherent cell, based scaffold, scaffold, 3d broccoli, scaffold production protocol, scaffold production protocol for more information, cell, scaffolds this protocol, isotropic arrangement of pore, pore,
Funders Acknowledgements:
Sigma Xi Grant in Aid of Research
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol describes how to seed and culture adherent cells on scaffolds derived from decellularized broccoli stalk tissue. It is part of a protocol collection describing how to produce and use the plant-based scaffolds for short-term (days), in vitro, cell-culture experiments. The scaffolds are composed of an isotropic arrangement of pores (median diameter, 70 μm), and have a stiffness in the range of mid-stiffness human soft tissues. Please see the scaffold production protocol for more information.
Materials
In addition to standard cell culture lab supplies and the reagents listed below, you will require:
- 96-well plates for culturing cells on the scaffolds
- Cell culture reagents appropriate for your cell type
For the proof of concept experiments in the associated manuscript, mouse embryonic fibroblasts (MEF) were cultured in DMEMGibco - Thermo Fisher ScientificCatalog #11995065 + 10 % volume Fetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #16140071 + 1 % volume Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 + 1 % volume MEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050 .
Protocol materials
Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144
human plasma fibronectinCorningCatalog #354008
Fetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #16140071
Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
MEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050
DMEMGibco - Thermo Fisher ScientificCatalog #11995065
Troubleshooting
Before start
This procedure was developed and tested with mouse embryonic fibroblasts. Your cell type may require optimization of seeding densities, incubation times, and culture conditions.
Prepare a 20 Mass Percent solution ofhuman plasma fibronectinCorningCatalog #354008 in sterile Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144 . Make 200 µL for each scaffold.
1m
Use forceps to transfer scaffolds into individual wells of a 96-well plate. Ensure that just enough force is used to grip the flat faces of each scaffold but not so much as to crush or otherwise damage the scaffold.
Note
Pouring the scaffolds into a sterile dish (e.g. a sterile Petri dish) can make it easier to pick them up and transfer them to the well plate, particularly if you need to transfer a large number of scaffolds or if the storage tube is long and narrow (e.g. a 15 ml conical tube).
5m
Add 200 µL sterile PBS to each well containing a scaffold.
2m
Aspirate the PBS from the wells.
Note
For this and all aspiration steps, avoid touching the scaffold with the aspirator tip so as to prevent damage to the scaffold. For 6.5 mm scaffolds, which completely occupy the bottom of the well (in a 96-well plate), tilt the well plate and aspirate down to the top surface of the scaffold but do not attempt to push the aspirator tip to the bottom of the well.
2m
Add 200 µL of the fibronectin solution to each well containing a scaffold.
2m
Incubate for 1 hour 37 °C in a cell culture incubator or seal the plate with parafilm and incubate overnight at4 °C .
1h
Aspirate the fibronectin solution.
2m
Add 200 µL sterile PBS to each scaffold well and incubate in the cell culture hood for 30 minutes.
32m
Meanwhile, trypsinize the cells, collect them as a suspension, and count them according to your lab's standard operating procedures.
30m
Aspirate PBS from the scaffolds.
2m
Seed desired number of cells onto the scaffolds in complete growth medium (or medium of choice). Each scaffold should have 100-200 μl total volume.
Note
The proof-of-concept experiments in the associated manuscript used mouse embyronic fibroblasts (MEFs) seeded at densities between 1,000 and 15,000 cells per 3.5 mm scaffold. To seed 1,000 cells per scaffold, we diluted the cell suspension to 5,000 cells/ml and pipetted 200 μl onto each scaffold.
5m
Culture according to cell requirements for at least two hours to allow the cells to fully attach before performing any manipulations (e.g. changing the medium). Refresh the cell culture medium every 48 h.