3D tissue scaffolds make better models for research and clinical use. In this study, the researchers decellularized apple slices. They grow NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts, and human HeLa epithelial cells into the scaffolds. The cells can grow and live for over 12 weeks. The cells were as dense as other popular scaffold materials. Since apples are cheap and renewable, this may be a great option for scaffolding.
2D cell cultures are not as good of a model for research as 3D models. Cells grown in 2D conditions will get flatter as time goes on, but can return to their original shape if they are grown in 3D conditions. Cellulose is the main reason scaffolds are possible. Cellulose is the carbohydrate that makes up the cell walls of plants. It is not easily digestible, and has shown to be useful in medicine. Cellulose can be used as dialysis tubing. 3D cell cultures have better cell to cell connections through gap junctions. Apple tissue is a good choice because it has lots of pores, so the cells will be able to transport nutrients easily. The apple must be decellularized, which means removing all proteins, lipids, and nucleic acids to leave a pure cellulose scaffold. This study looks at mouse NIH3T3 fibroblasts, mouse C2C12 myoblasts, and human HeLa epithelial cells in the apple scaffold. They looked at the structure, chemical properties, and mechanical properties of the scaffolds.
Materials and Methods
Apple Tissue Preparation, Decellularization, and Storage
McIntosh Red apples were used for this study. The apples were sliced, sanitized, and submerged in sodium dodecyl sulphate (SDS). SDS is a detergent that will dissolve everything except for the cellulose, effectively decellularizing the apples. The scaffolds were treated with antibiotics and a buffer.
The scaffolds were either treated with nothing, collagen, or glutaraldehyde. The three types created were called native, collagen coated, or cross-linked.
C2C12 mouse myoblasts, NIH3T3 mouse fibroblasts, and HeLa human epithelial cells were separately given to different scaffold types. The media used for this study was standard mammalian media (DMEM) supplemented with 10% fetal bovine serum, antibiotics, and buffer.
In Vitro Culture in Cellulose Scaffolds
The scaffolds were placed into a 24-well tissue culture plates and a droplet containing 6,000,000 cells was given to each scaffold. They were grown in cell media for the next 12 weeks.
Immunohistochemistry techniques were used to stain the cells.
Confocal Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy were all used to image the samples.
Preparation of Cellulose Scaffolds
The apple tissue made for a very porous sponge-like structure after decellularization. The other plant cells, proteins, and nucleic acids were successfully removed because only the cellulose scaffold was visible on Scanning Electron Microscopy.
Mechanical Properties of Native and Modified Cellulose Scaffolds
The scaffolds were treated with collagen or glutaraldehyde to make samples called collagen and cross-linked scaffolds. They used Atomic Force Microscopy to measure the elasticity of four samples: the untreated apple tissue, native decellularized scaffold, collagen scaffold, and cross-linked scaffold. The elasticity of the collagen scaffold and cross-linked scaffold was much higher than the other two samples. This shows that apple scaffolds could mimic mammalian tissues after treatment with certain agents.
Mammalian Cell Culture in Native, Collagen Functionalized, and Chemically Cross Linked Cellulose Scaffolds
The C2C12 myoblast, NIH3t3 fibroblast, and HeLa epithelial cells were all grown separately in the native, cellulose, and cross-linked scaffolds. All cells grew at a rapid rate, called proliferation. The cells were growing all throughout the scaffold, from the surface to deep within the cellulose. The different combinations were also tested for actin stress fibers. They all tested positive for actin stress fibers. This means the the cells were strongly attached to the scaffold, not just physically enclosed in the scaffold.
Proliferation and Viability
To see how long the cells could live in the scaffold, the researchers took imaging at 1, 8, and 12 weeks. The HeLs and C2C12 cells grew two times faster than the NIH3T3. Then, the researchers stained all of the dying or dead cells a different color from all of the healthy cells. About 98% of the cells were living and healthy after 12 weeks, showing that this scaffold could be maintained long term.
Cells grown in 3D have a lot of differences than those grown on a 2D surface. 3D scaffolds allow cells to have proper shape, communication, and growth. Two types of scaffolds exist: artificial and decellularized scaffolds. Artificial scaffolds allow the research team to have more control over the shape, size and other properties of the scaffold because they can synthesize it from scratch. Decellularization can produce a natural and cheap scaffold that is much easier to make. Apple tissue is a great option for decellularization. Apple tissue is naturally porous and easily decellularized. All cell types in this study could easily adhere to the scaffold and grow. Nutrients could be exchanged all the way to the middle of the scaffold in still standing culture media, so no current was needed to penetrate the scaffold. Some cell types grow better in certain elasticity conditions, so tuning the cellulose scaffold with collagen or glutaraldehyde is a great way to make cellulose a favorable scaffolding material for several cell types. This is also supported by the stress actin fibers that formed, showing good levels of adhesion to the scaffold for all cell types. The amount of dying cells was very small in the 3D scaffold as well. Porous scaffolds have benefits and challenges. Cells will not adhere as well to a scaffold with too many holes. This was the case in the apple scaffold, so only 2,000,000 of the 6,000,000 cells they seeded actually attached to the scaffold. A porous scaffold does allow dead cells to be washed out easily. Finally, nutrients and cell communication is present in a porous scaffold. Combined with the cheap price and easy decellularization process, apple scaffolds may prove to be very effective for research and clinical treatment options.