Team:BUCT-China/Proof Of Concept

Result
Cell result
3D printing

Proof of Concept

The experimental design of 3D printing scaffold and cell culture, as well as the production of raw material PHFA and collagen for scaffold are all illustrated in Design page as follow:
https://2021.igem.org/Team:BUCT-China/Design
The results of experiments regarding to raw material PHFA and collagen are demonstrated in Engineering page as follow:
https://2021.igem.org/Team:BUCT-China/Engineering

I.Basic scaffold result

1. Rheological test(ratio is sodium alginate: gelatin)

Since sodium alginate significantly improves the viscosity of the overall system, 3% (W/V) was selected according to literature review, and the viscosity was adjusted by adjusting the gelatin concentration.

According to the chart, when the gelatin concentration is 10%, 15% and 20%, the viscosity is low and cannot meet the printing support solid scaffold demand. The viscosity of 25% and 30% has little difference, and 25% has met the printing demand. According to the principle of economy, 25% is selected as the proportion for subsequent experiments.

2. Printing experiment (operation is carried out according to the above scheme)

(1) Printing with the ratio of 3:20
Conditions: At room temperature, 40Kpa pressure, 410um for sprinkler head,20mm×20mm×4mm for G code, 0.4mm for a layer, 0°/90° for scaffold type.

Discussion: The prototype was found, but it was also proved that when the gelatin concentration was low, both the material's own viscosity support and low temperature setting effect was poor. The gelatin proportion should be increased (figure 1. A)

(2) Printing with a ratio of 3:25
Conditions: the wall temperature is 37℃, the air pressure is about 38kpa, the sprinkler head is 410um, the G code is 20mm×20mm×4mm, a layer of 0.4mm, the filling rate is 15%, the type is 0°/90°, and the calcium chloride cross-linking is quickly finalized after completion.

Discussion: Preliminary scaffold design has been completed which can maintain a certain thickness and aperture, and the printing conditions are mild, so it can be used for mixed printing with cells. (figure 1. B)

(3) Explore the use of different printing models
Conditions: the proportion of mixed material is 3:25, the wall temperature is 37℃, the air pressure is about 40kpa, the sprinkler head is 410um, the rectangular model of 20mm×20mm×4mm is used, a layer of 0.4mm, the filling rate is 15%, the type of pore of the support is 45°/135°, and the crosslinking of calcium chloride is completed quickly.

Conditions: the proportion of mixed material is 3:25, the wall temperature is 37℃, the air pressure is about 40kpa, the sprinkler head is 410um, the R=12mm, H=4mm model, a layer of 0.4mm, the filling rate is 15%, the type of pore of the support is 45°/135°, and the crosslinking of calcium chloride is completed quickly.

Discussion: After the model is improved, the scaffold has good molding effect, the overall appearance is more beautiful, more uniform, the pore size is moderate, and the printing conditions are mild, which can be used for mixed printing with cells. (figure 1. C,D)

Figure.1

A) Printing with the ratio of 3:20
B) Printing with a ratio of 3:25
C,D) the use of different printing models
E) 3D bioprinter
F) Process of 3D bioprinting.

(4) Parallel printing of the scaffolds
After we identify the printing conditions and methods, we start parallel printing of the same type of scaffold for subsequent control tests(figure.2A), and preserve it in calcium chloride solution at the same time (figure.2B)，and take part of the printed scaffold to freeze-dry for porosity detection.(figure.2C)

(5)stability testes
We put the printed scaffold into PBS solution. We find the scaffold will not collapse in cell culture medium, but can still maintain a certain aperture and shape, with good stability, and can be used for cell growth and differentiation for a certain period.(figure.2D)

Figure.2

A) Parallel printing of the same kind of scaffold
B) The preservation of scaffold in calcium chloride solution
C) The freeze-dried hydrogel
D) THE comparison of the first and fifth days in PBS solution

(6)Determination of porosity

According to the experimental results, the porosity of scaffolds is about 80%-95%. As three-dimensional porous scaffolds, their porosity is relatively large, which can meet the requirements of nutrient transport and metabolite discharge required by cells.

II. Cell result

1. By looking at the cell state, we found that muscle satellite cells grow best when they are transferred to the third generation. The figure shows P3 chicken muscle satellite cells successfully extracted by enzyme digestion of chicken muscle tissue. Looking at the diagram, we can see that the cells are growing vigorously and neatly, with few impurities.

2. Draw the cell growth curve

The cell curve was drawn by cell counting every three days. It could be seen that the number of cells changed from the 12th day and increased significantly after the 15th day. The cell growth rate reached its maximum during the period of 21-24 days.

III. 3D printing of cells and scaffolds

Figure.3

A) Overall view of scaffold cell mixture
B) Detail view of the scaffold cell mixture

Above is the picture of cells and scaffolds printed together and incubated in an incubator for one day.

First, by observing the shape of the scaffold, it was found that the scaffold did not collapse significantly, indicating that its support strength was enough to allow cells to grow on it.

We are committed to proving that PHFA and collagen can be used as scaffold materials for tissue engineering. We tested cytotoxicity in two ways. PHFA and collagen have positive effects on cell growth.

In order to detect cytotoxicity quickly, sensitively and objectively, we first used neutral red dye kits. Neutral red staining works by drawing reactive dyes into the lysosomes of undamaged cells. Since only living cells absorb neutral red dye and thus enter the lysosomes of cells, the more toxic the material, the fewer living cells and the less dye can be absorbed. As can be seen from the figure above, the bracket is dark and evenly distributed. By visual observation, we could confirm that the cells survived in large numbers and were evenly mixed with the scaffold. These results indicate that PHFA and collagen scaffold have positive effects on cell growth.

To further verify cytotoxicity. We also used the Calcein-AM/PI Double Stain Kit for 2D and 3D observation of scaffold and cell mixture through confocal microscopy.

Calcein-AM is a cell staining reagent that can fluorescence-label living cells. Due to enhanced hydrophobicity on the basis of Calcein, Calcein-AM can easily penetrate living cell membranes. When it enters the cytoplasm, esterase will hydrolyze it into Calcein and leave it in the cell, emitting strong green fluorescence.

PI, a nuclear dye, cannot pass through the membrane of living cells. It passes through the disordered regions of dead membranes to reach the nucleus, where it inserts into the DNA double helix of cells and produces red fluorescence.

Figure.4

A) B) 2D-fluorescence images (green: live cells and red: dead cells)
C) 3D-fluorescence image(green: live cells and red: dead cells)

In A) B) The light gray part is the gap of the support, the dark gray part is the main body of the support, and the point pointed by the arrow is the edge of the support material. It can be clearly seen that all the cells are distributed on the main body of the scaffold, and the cells are closely combined with the scaffold and evenly distributed. Living cells are green and dead cells are red. 2D confocal microscopy showed that the number of labeled living cells accounted for a large proportion, while the number of dead cells was small. The results showed that PFHA and collagen mixed scaffold had positive effect on cell growth.

The cell morphology obtained in 3D culture (Fig.4 C)) is more accurate than in 2D, and the effect on cell function is more similar to that of cells in vivo. Z-axis scanning with confocal laser microscope and observation after 3 D construction, the results are shown in the figure. Due to the existence of spontaneous fluorescence in the material, green is the labeled living cells, red is the material, and there are fewer labeled dead cells.

Cells cultured in 3D were found to be more viable under confocal microscopy. It is further proved that PHFA and collagen are very feasible as scaffold materials.

So far, our team completed the mixed printing and co-culture of cells and scaffolds, which confirmed the feasibility of cultured meat. In the coming years, we'll be working on getting cells to grow and differentiate better on the scaffold, and eventually make real cultured meat.