Decellularization of placentas: establishing a protocol

Abstract

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Figure 1.. Macroscopic and microscopic aspects of fetal (A–F) and maternal (G and H) portions of canine placenta subjected to decellularization. Perfusion (A, C, E and F) and immersion (B, D) techniques in the fetal portion of placenta led to cell removal and samples with gelatinous and translucent aspects. No apparent differences regarding the percentage of remaining cells were found in placentas frozen at –20°C (C, E) and –80°C (D, F) prior to decellularization. Perfused placentas did not decrease the incubation time and frozen placentas required more time of incubation with decellularization reagents to remove cells. The application of 1% sodium dodecyl sulfate detergent (G) and its association with 1% Triton X-100 (H) were ineffective to completely remove cells (black arrows) from the maternal portion of placenta. A and B: Scale bar = 1 cm. C-H: HE staining; 20×; scale bar = 100 µm.

Figure 2.. Macroscopic and microscopic aspects of fetal portions of canine placenta. Left column (control group), middle column (Protocol I – 5 mM EDTA + 50 mM TRIS + 0.5% antibiotic) and right column (Protocol II – 5 mM EDTA + 0.05% trypsin + 0.5% antibiotic). Protocol I led to better conservation of the macroscopic and microscopic structures. In E (upper left corner, 20×; scale bar = 100 µm) it is possible to observe a structurally organized extracellular matrix (ECM), but with nuclei of remaining cells (black arrow). Vascular wall architecture (red arrow) was preserved (B, H, and K). Protocol II was more aggressive to ECM as shown in F (upper left corner, 20×; scale bar = 100 µm), but was more effective in removing cells. A-C: macroscopic aspect, scale bar = 1cm; D-F: HE staining, 40×, scale bar = 50 µm; G-I: Masson's trichrome staining, 20×, scale bar =100 µm; J-L: fibrillar collagen network under polarized light, Picrosirius red staining, objective 16, magnification 1.25, scale bar = 20 µm.

Figure 3.. Macroscopic and microscopic aspects of maternal portions of canine placenta. Left column (control group), middle column (Protocol I - 5 mM EDTA + 50 mM TRIS + 0.5% antibiotic) and right column (Protocol II - 5 mM EDTA + 0.05% trypsin + 0.5% antibiotic). Macroscopic changes were not observed when Protocols I (B) and II (C) were compared. In Protocol I (E) cell content was not eliminated completely (black arrow). Samples subjected to Protocol II showed less cells and ECM derangement (F). Blood vessels structure (red arrow) were more preserved in Protocol I (H). A–C, macroscopic aspect, scale bar = 1 cm; D–F, HE staining, 40×, scale bar = 50 µm; G–I, Masson's trichrome staining, 20×, scale bar = 100 µm; J–L, fibrillar collagen network under polarized light, Picrosirius red staining; objective 16; magnification 1.25; scale bar = 20 µm.

Figure 4.. Scanning electron microscopy of fetal (A–C) and maternal (D–F) portions of canine placenta. Left column (control group), middle column (Protocol I – 5 mM EDTA + 50 mM TRIS + 0.5% antibiotic) and right column (Protocol II – 5 mM EDTA + 0.05% trypsin + 0.5% antibiotic). The red asterisks indicate the spaces that resemble pores observed mainly in fetal portions decellularized with Protocol I (B). Scale bar = 3 µm.

Figure 5.. DNA quantification performed using Quant-iTTMPicoGreen^®dsDNA reagent. Samples subjected to Protocol II (EDTA + 0.05% trypsin + 0.5% antibiotic) showed a trend to less concentration of DNA content compared to samples processed according to Protocol I (5 mM EDTA + 50 mM TRIS + 0.5% antibiotic), but the values were not statistically significant (P>0.05). Data are reported as means±SD (Protocol I: 891±690.7071 for maternal portion and 1125±787.0756 for fetal portion. Protocol II: 412±365.9669 for maternal portion and 418±373.6606 for fetal portion).

Figure 6.. Immunofluorescence of extracellular matrix proteins from fetal portions of canine placenta in control group (A–D), Protocol I (5 mM EDTA + 50 mM TRIS + 0.5% antibiotic) (E–H), and Protocol II (5 mM EDTA + 0.05% trypsin + 0.5% antibiotic) (I–L). Laminin, fibronectin, collagen types I and III were expressed in control groups (A–D); collagen type III was less evident in the same samples of decellularized groups (E–L). Scale bar = 40 µm.

Figure 7.. Immunofluorescence of extracellular matrix proteins from maternal portions of canine placenta in control group (A–D), Protocol I (5 mM EDTA + 50 mM TRIS + 0.5% antibiotic) (E–H), and Protocol II (5 mM EDTA + 0.05% trypsin + 0.5% antibiotic) (I–L). Laminin, fibronectin, collagen types I and III were expressed in control groups (A–D); collagen type III was less evident (D). In decellularized groups, collagen type III was not identified in maternal portions in both protocols (H, L). Other proteins were observed in all samples after Protocols I (E–G) and II (I–K). Nuclear staining with DAPI was absent, indicating cell removal in decellularized groups (E–L). Scale bar = 40 µm.