The culture of one cell type (monoculture) presents some limitations, since the interaction with other cell types cannot be considered. Consequently, the monoculture model does not mimic the real multicellular interaction during the progression of the disease. Simultaneous co-culture is a system where two cell types are seeded together and allows the study of cell-to-cell interactions. This approach reproduces more accurately cell connections observed in the in
vivo microenvironment. The development of more complex and sophisticated co-culture models,
can reproduce more accurately the intercellular interactions and the biochemical response. To investigate whether HUVEC, stimulated with anti-VEGF drugs, have protective effects against oxidative stress on RPE, and can have effects on NO release, proliferation, migration, viability, apoptosis and mitochondrial membrane potential, we co-cultured RPE with HUVEC by using Transwell inserts (Life Science; Gerenzano, Milan, Italy, and ACEA Bioscience) (Figure 7). Experiments were performed both in physiological and peroxidative conditions. Cell and tissue culture techniques are becoming increasingly important for basic and applied life science research. The development of new culture vessels and cell attachment substrates is currently being driven by the need to produce an environment that resembles the in vivo state as closely as possible to enable the growth of specialized cell types. Consequently, using permeable supports with microporous membranes has become a standard method for culturing these cells. These permeable supports have allowed significant improvements in culturing polarized cells since these permeable supports permit cells to uptake and secrete molecules on both their basal and apical surfaces and thereby carry out metabolic activities in a more natural fashion. In Table 5, the characteristics of the Transwell inserts which were used, are shown.
Materials and methods
Multiple well plate 96-well insert 24-well insert 6-well insert E-plate insert 16
Company Life Science Life Science Life Science ACEA Biosciences
Membrane material polyester polyester polyester polyester
Membrane thickness 10 µm 10 µm 10 µm 10 µm
Collagene treated no no no no
Cell visibility good good good good
Optical properties clear clear clear clear
Transwell insert diameter 4.26 mm 6.5 mm 24 mm 4.26 mm
Insert membrane growth area 0.143 cm2 0.33 cm2 4.67 cm2 0.143 cm2
Pore size 0.4 µm 0.4 µm 0.4 µm 0.4 µm
Pore density 4*106 pores/cm2 4*106 pores/cm2 4*106 pores/cm2 4*106 pores/cm2
Volume added per plate well 0.235 ml 0.6 ml 2.6 ml 0.235 ml
Volume added to inside of
Transwell insert 0.075 ml 0.1 ml 1.5 ml 0.075 ml
Table 5: Characteristics of Transwell inserts
Figure 7: In A, an example of E-Plate Insert in combination with the E-Plate 16. In B, an example of Insert in
combination with the 24-well plate
For co-culture experiments, HUVEC were plated in the apical compartment of the insert while, RPE cells, were plated in the basal compartment. Transwell inserts were used by first adding medium to the multi-well plate, followed by adding the Transwell inserts, and lastly adding the medium and cells to the inside compartment. An initial equilibrium period may be used to improve cell attachment. The plate should then be incubated for at least one hour at the same temperature that will be used to grow the cells. The cells are then added in fresh medium to the Transwell insert and returned to the incubator over-night. Next day, HUVEC were stimulated with Aflibercept
id
Lid E-Plate Insert
E-Plate 16 24-well plate
Materials and methods
(0.025 mg/ml and 0.5 mg/ml, for 30 min) or Ranibizumab (0.025 mg/ml and 0.2 mg/ml, for 30 min). In addition, in some experiments, Aflibercept (0.5 mg/ml) or Ranibizumab (0.2 mg/ml) were also given after pre-treatment with the NOS inhibitor, L-NAME (4 µM, for 15 min; Sigma), the p38 MAPK inhibitor, SB203580 (4 µM, for 30 min; Sigma), the phosphatidylinositol 3′-kinase (PI3K) inhibitor, wortmannin (4 µM, for 15 min; Sigma), or the MAPK/ERK inhibitor, UO126 (4 µM, for 15 min; Sigma). After the treatment, medium was changed and new completed fresh medium was added for 24 h, in order to give the HUVEC time to induce any responses from the RPE cells. Next day, using a sterile forceps, all Transwell inserts were removed, and some samples of RPE cells were treated with 200 µM H2O2, in order to induce oxidative stress. At the end of
stimulations, various assays were performed as described above (Figure 8).
DAY 1: PLATE CELLS AND LEAVE THEM IN INCUBATOR OVER NIGHT 37°C, 5% CO2
Figure 8: Schematic representation of experimental models, is shown
3.3.1 Evaluation of NO release, cell viability, mitochondrial membrane potential and ROS production in RPE cells
NO release (Griess assay), cell viability (MTT assay), mitochondrial membrane potential (JC-1 assay), and ROS production (DCFDA-Cellular ROS Detection Assay Kit), were measured in RPE, co-cultured with HUVEC, by following the manufacturer´s instructions as described in paragraphs
COMPLETE MEDIUM HUVEC CELL LINE
MICROPOROUS MEMBRANE INCUBATOR FOR 1 H MICROPOROUS MEMBRANE
ARPE-19 CELL LINE ARPE-19 CELL LINE
DAY 2: HUVEC ARE STIMULATED WITH ANTI-VEGF (FOR 30 MIN) DRUGS/INHIBITORS (FOR 15 MIN) AND THEN, THE CULTURE MEDIUM OF HUVEC IS CHANGED
DAY 3: AFTER 24 H, TRANSWELL INSERTS ARE REMOVED AND ARPE-19 ARE STIMULATED WITH H2O2.
THE SPECIFIC ASSAY IS PERFORMED
Aflibercept Ranibizumab
200 µM H2O2
HUVEC CELL LINE
INCUBATOR FOR 24 H
ASSAY
MICROPOROUS MEMBRANE
Materials and methods
3.2.2, 3.2.3, 3.2.4, and 3.2.7. For co-culture, HUVEC (7500 cells/insert in 96-Transwells plate for Griess assay, and 10 000 cells/insert in 96-Transwell plate for MTT and JC-1 methods), were stimulated with Aflibercept (0.025 mg/ml and 0.5 mg/ml, for 30 min) or Ranibizumab (0.025 mg/ml and 0.2 mg/ml, for 30 min). Another pool of cells was treated with Aflibercept (0.5 mg/ml, for 30 min) and Ranibizumab (0.2 mg/ml, for 30 min), after pre-treatment with various inhibitors, as described above. After stimulations, medium was changed and new F-12K medium was added for 24 h. Next day, each Transwell insert was removed and some RPE samples (7500 cells/well in 96-Transwell plate for Griess assay and 10 000 cells/well in 96-Transwell plate for MTT and JC-1 assays) were treated with for 30 min, in order to induce oxidative stress (Figure 9). Finally, the absorbance (as specified in previous paragraphs) was read by using a spectrophotometer (VICTOR™ X Multilabel Plate Reader; PerkinElmer).
For ROS production (25 000 HUVEC cells/insert in 96-Transwell plate and 25 000 RPE cells/well in 96-Transwell plate), the same experimental protocol was used but in the absence of inhibitors (Figure 10). Experiments were conducted in triplicate and repeated at least five times.
Figure 9: The Flowchart shows a schematic representation about the co-culture experiments performed by
using Griess, MTT and JC-1 methods
Figure 10: The Flowchart shows a schematic representation about the co-culture experiments performed