PLGA Viability

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Poly(lactic-co-glycolic acid), or PLGA, is a common thermoplastic used in bioprinting for its tunable degradable and mechanical properties[1,2]. The degradation and mechanical properties of this material can be altered by adjusting the ratios of the copolymers polylactide and polyglycolide [1,2].

Thermoplastics in bioprinting are often used as a support structure to provide extra mechanical strength and as reinforcement for matrix bioinks[1,2]. Polycaprolactone is a biodegradable thermoplastic used in a variety of medical applications, including bioprinting. This study tested the viability and print resolution of BioPCL, a polycaprolactone based bioink, with the BioBot 1.

Results

Viability Data

ATP of thin films demonstrates an increase in ATP from day 1 to day 3, with statistically higher ATP values on day 3 and day 7 when compared to day 1. Likewise, LIVE/DEAD images confirm viable cultures of HNDFs seeded on PLGA thin films after 1,3 and 7 days of culture.

ATP live dead PLGA updated

Figure 3: ATP results (A) demonstrate that PLGA is able to support viable cell cultures. (*) indicates statistical difference. LIVE/DEAD images (B) also confirm viable cell cultures seeded on PLGA at days 1,3 and 7.

These results demonstrate the ability of PLGA to support viable cultures and create consistent, high resolution 3D geometries. This support material can be used in combination with matrix bioinks or on its own to create 3D culture environments for tissue engineering.

Print Resolution 

resolution PLGA

Figure 1: Images of printed structures demonstrate reproducibility and high resolution. Left: image of line created through the “string” printing method. Center: image of printed lattice, cylinder and line structures. Right: Brightfield image of printed lines at 2.5x magnification (scale bar 0.5 mm).

PLGA was printed using print parameters and settings provided by BioBots (Figure 1). For traditional printing methods, pore width average was 0.45 ± 0.20 mm, while line width average was 0.51 ± 0.14 mm (Table 1). For string printing methods, line width average was 0.14 ± 0.47 mm (Table 1).

PLGA Line Resolution
Print Method Needle Gauge (mm) Resolution (mm)

Traditional

25(0.43) 0.51 ± 0.14

String

23 (0.56)  0.14 ± 0.47

Table 1: Resolution for traditional and string printing methods with PLGA.

Methods

Cell Culture

Primary Human Neonatal Dermal Fibroblasts (HNDFs) from ATCC were cultured at 37 °C and 5% of CO2. HNDFs were cultured using Dulbecco’s Modified Eagle Medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin-amphotericin (Corning). Passage numbers under 10 were used.

Thin Film Fabrication

Viability of PLGA was tested with cell seeding of human neonatal dermal fibroblasts. First, PLGA was sterilized with UV light over night. Then, thin films were fabricated by melting PLGA at 100°C on a glass coverslip, then flattening with a second glass coverslip. Thin films were then allowed to cool to room temperature before removal from coverslips.

HNDFs were suspended at a concentration of 9500 cells mL-1 and pipetted into the wells containing the PLGA thin films. Enough cell solution was used to submerge the samples. After overnight incubation, media was exchanged.

Viability Assessment

To quantitatively assess cell viability, CellTiter-Glo 3D ATP Assay (Promega) was performed on days 1,3 and 7 of culture according to manufacturer’s protocol and analyzed using a BioTek Synergy 2 Plate Reader. A LIVE/DEAD kit (Life Technologies) was used to qualitatively assess viability of samples.

Statistical analysis was performed using an Analysis of Variance (ANOVA) test to determine if differences were present amongst treatment groups. If differences were determined from the ANOVA, a post hoc Tukey’s multiple comparison test was used to determine statistical differences between groups tested. A confidence level of 95% (α=0.05) was used for all analyses. Error bars on graphs represent the standard deviation from the mean.

Printing 

PLGA was printed using print parameters and settings provided by BioBots. Line width and pore size were analyzed with ImageJ software. Lattice and line designs were each printed 3 separate times. 3 images of each print were taken, then three measurements were taken from each image.

Traditional printing methods were tested with a line and a lattice print design. String printing methods, also described in settings provided by BioBots, were tested with a star design.

References

[1]
Pati, Falguni et al, “Ornamenting 3D Printed Scaffolds with Cell-laid Extracellular Matrix for Bone Tissue Regeneration Biomaterials, 2015(37), pp. 230 – 241.
[2]
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