Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Abstract

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces. Correspondence

Biodegradable polymer blends: miscibility, physicochemical properties and biological response of scaffolds

“…Cells adopted a round morphology with thin and long microvilli (Fig. (A)) in the porous area of PLLA, whereas in the PLLA non‐porous areas the cells were flattened with filaments . Round morphology and filaments are indicative of spreading cells with their thin cytoplasmatic filaments.…”

“…Changes in Vero cell (kidney cell line derived from the African green monkey) morphology and cellular differentiation pattern were noted depending on blend proportions possibly due to factors such as polymer surface energy, roughness, rigidity and surface tension (Fig. ) . Cells growing on porous or non‐porous areas in PLLA had different morphologies.…”

Bioresorbable Polymers for Tissue Engineering

“…We showed that Vero cells produce extracellular matrix rich in fibronectin and collagen when cultured on dense or porous PLLA membranes, PHBV scaffolds or PLLA/PHBV blends of different proportions [21,25]. This finding may explain the observation of a significant proliferation rate despite the initially slow cell adhesion to these scaffolds [25,52].…”

Cell culture on PCL/PLGA blends

“…18 Previously, the sterilized blends (n ¼ 6) were placed in a 96-well plate (Corning, USA) with 100 lL of culture medium and incubated at 37 C for 24 h. After incubation, 2 Â 10 5 cells/mL in 100 lL DMEM medium supplemented with 10% FBS were added to the wells containing the membranes. The cells were cultured for 2 and 24 h to allow cell adhesion and to conduct direct cell cytotoxicity assays, respectively.…”

“…The cells were cultured for 2 and 24 h to allow cell adhesion and to conduct direct cell cytotoxicity assays, respectively. After the cells were washed twice with 0.1M phosphate-buffered saline (PBS), pH 7.4, at 37 C and incubated with 100 lL DMEM medium, a MTT assay mixture [10 lL per well, containing 5mg/mL of 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Sigma] was added to each well and incubated for 4 h at 37 C. After 4 h, 100 lL of dimethyl sulfoxide (DMSO, Sigma) and 25 lL of glycine/Sorensen buffered solution replaced the assay mixture in each well to dissolve the formazan crystals, according to Santos et al 18 Absorbance was quantified by a spectrophotometer at 540 nm, using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA). MTT is a colorless tetrazolium salt that forms a dark compound when oxidized by mitochondria, which is detected by spectrophotometer.…”