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. 2019 Jul 2;12(13):2125.
doi: 10.3390/ma12132125.

Real-Time Live-Cell Imaging Technology Enables High-Throughput Screening to Verify in Vitro Biocompatibility of 3D Printed Materials

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Free PMC article

Real-Time Live-Cell Imaging Technology Enables High-Throughput Screening to Verify in Vitro Biocompatibility of 3D Printed Materials

Ina G Siller et al. Materials (Basel). .
Free PMC article

Abstract

With growing advances in three-dimensional (3D) printing technology, the availability and diversity of printing materials has rapidly increased over the last years. 3D printing has quickly become a useful tool for biomedical and various laboratory applications, offering a tremendous potential for efficiently fabricating complex devices in a short period of time. However, there still remains a lack of information regarding the impact of printing materials and post-processing techniques on cell behavior. This study introduces real-time live-cell imaging technology as a fast, user-friendly, and high-throughput screening strategy to verify the in vitro biocompatibility of 3D printed materials. Polyacrylate-based photopolymer material was printed using high-resolution 3D printing techniques, post-processed using three different procedures, and then analyzed with respect to its effects on cell viability, apoptosis, and necrosis of adipogenic mesenchymal stem cells (MSCs). When using ethanol for the post-processing procedure and disinfection, no significant effects on MSCs could be detected. For the analyses a novel image-based live-cell analysis system was compared against a biochemical-based standard plate reader assay and traditional flow cytometry. This comparison illustrates the superiority of using image-based detection of in vitro biocompatibility with respect to analysis time, usability, and scientific outcome.

Keywords: 3D printing; adipogenic mesenchymal stem cells; biocompatibility; flow cytometry; in vitro study; real-time live-cell imaging technology.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow chart of performed experiments. The in vitro biocompatibility of 3D printed material was evaluated using three different approaches.
Figure 2
Figure 2
Schematic process of 3D printing, post-processing and extraction medium preparation. Cleaning steps (1): freezing of 3D printed objects (15 min, −18 °C), heat steam in a water bath (45 min, 65 °C), ultrasonic bath with detergent (30 min, 65 °C). Sterilization steps (2): disinfection in ethanol (70%, v/v, 1 h, RT) or sodium hypochlorite (2%, v/v, 1 h, RT) or UV light exposure (1 h, RT). Biocompatibility testing steps (4) then followed an incubation of 3D printed objects in cell culture medium according to EN ISO 10993-12 (2012) (3). (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v) and EM 3: EM obtained by incubation of 3D printed material sterilized by UV light.
Figure 3
Figure 3
Schematic illustration of the working process of real-time live-cell analysis. (A) Placing of the real-time live-cell imaging system inside a cell culture incubator; (B) automatically acquire images over time; (C) receive images of all locations in the culture vessel at once; (D) imaging masks identify regions of interest; and (E) the results can be monitored in real-time and (F) display quantitative and kinetic analyses of all culture vessels at once.
Figure 4
Figure 4
Results of CellTiter-Blue® cell viability assay (CTB assay) to analyze the metabolic capacity (shown as cell viability in %) of MSCs. (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v). EM 3: EM obtained by incubation of 3D printed material sterilized by UV light. All experiments were repeated several times (n = 13) and compared to MSC cultivation in regular cell culture medium (Control).
Figure 5
Figure 5
Results of flow cytometric studies on apoptosis and necrosis of MSCs over a period of 30 h. The percentage of living, apoptotic and necrotic cells are analyzed per cultivation. A caspase 3/7 signal (green) represents apoptotic cells; the cytotox-signal (red) is correlated to necrotic cells. (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v). The experiments are compared to MSC cultivation in regular cell culture medium (Control) and were performed three times (n = 3).
Figure 6
Figure 6
Cell growth of MSCs determined by cell counting using Trypan blue staining. (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v). The experiments are compared to MSC cultivation in regular cell culture medium (Control) and were performed three times (n = 3).
Figure 7
Figure 7
Fluorescence images of MSCs over time by image-based live-cell analysis system (IncuCyte). Green fluorescence is related to apoptotic cells; red fluorescence shows necrotic cells. (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v). The experiments are compared to MSC cultivation in regular cell culture medium (Control) and were performed three times (n = 3). Cisplatin 50 µm: Positive control for apoptosis.
Figure 8
Figure 8
Analysis of cytotoxicity of the 3D printing polyacrylic material and apoptosis of MSCs by an image-based live-cell analysis system (IncuCyte). Cell growth, apoptosis and necrosis of MSCs are analyzed per cultivation. A caspase 3/7 signal (green) represents apoptotic cells; the cytotox-signal (red) is correlated to necrotic cells. (EM = extraction medium). EM 1: EM obtained by incubation of 3D printed material treated with ethanol (70%, v/v) in a disinfection process. EM 2: EM obtained by incubation of 3D printed material treated with sodium hypochlorite (2%, v/v). The experiments are compared to MSC cultivation in regular cell culture medium (Control) and were performed eighteen times (n = 18).

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