ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
ObjectiveTo investigate the growth characteristics of pancreatic cancer cells in the twodimensional culture system (monolayer) and threedimensional culture system (type Ⅰ collagen and extracellular matrix gel). MethodsThree pancreatic cancer cell lines (SW1990, PCT, and ASPC1) were cultured in monolayer, type Ⅰ collagen, and extracellular matrix gel, respectively. The growth patterns were observed, growth curves were detected by CCK8 test, and the cell cycle distributions were analyzed by propidium iodide staining. Results In the twodimensional culture system, cells grew in monolayer. In the type Ⅰ collagen and the ECM gel threedimensional culture system, cells formed multicellular spheroids (MCS), of which the growth rates were slower than those of the cells in monolayer. The proportions of S phase of SW1990, PCT, and ASPC1 cells in twodimensional culture system were significantly more than those in the type Ⅰ collagen on 4 d and 8 d 〔(29.6±3.0)% vs. (18.2±5.1)%, (33.6±2.1)% vs. (14.5±3.2)%, (33.1±1.8)% vs. (24.7±2.6)%; Plt;0.05〕, while the difference of proportion of three cell lines in G2/M phase was not different between twodimensional culture system and type Ⅰ collagen (Pgt;0.05). The proportions of G0/G1 phase of SW1990 and PCT cells cultured in the type Ⅰ collagen on 4 d and 8 d and ASPC1 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in twodimensional culture system (Plt;0.05). The proportions of S phase of ASPC1 cells and SW1990 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in the type Ⅰ collagen on 8 d (Plt;0.05). ConclusionsThe characteristics of pancreatic cancer cells in twodimensional and threedimensional culture systems are different. MCS culture system can better mimic the in vivo growth environment of cells in tumors.
Object ive To explore a method to recons t ruct eccr ine sweat gland- l ike s t ructure in vitro. Methods Isolated from the normal axillary full-thickness skin donated by volunteers sweat gland epithel ial cells were cultured in vitro and were observed under inverted phase contrast microscope. These cells at the density of 2 × 105/cm2 were inoculated underneath the Matrigel (group A), on the top of the Matrigel (group B) and in the Matrigel (group C), respectively,for three-dimensional culture. The formation of eccrine sweat gland-l ike structure was observed by confocal laser scanning microscope, HE staining and immunohistochemistry staining. Results Primary epithel ial cells in the secretory portion of sweat gland were attached and spindle-shaped 24 hours after inoculation, and were under polyclonal grain-l ike growth 2-3 days thereafter. Cobblestone-l ike appearances of these cells were evident 14 days after inoculation and the confluent cells were flat and polygonal with relatively big round cell nucleus. Morphologically, subcultured cells at passage 1 were similar to the primary cells; cells at passage 2 were irregular and most of them had long pseudopodium; cells at passage 3 were star-shaped and big and had fusion with adjacent cells. For group A, tubular structure was formed 11 days after three-dimensional culture. For group B, stretched and filamentous-shaped cytoplasm was observed 8 hours after three-dimensional culture, with the formation of lumen or half-lumen structure, but no significant prol iferation was evident. For group C, cell division and prol iferation occurred 2-3 days after three-dimensional culture; the prol iferated cells were closely arranged into tubular structure with obvious lacunae in the middle, which gradually developed into irregular ball-shaped structure with the increase of neonatal cells. The laser scanning confocal microscope observation showed the formation of spherical structure in group C, with tubular structure in the center of cell mass; HE staining testified the spherical structure in group C was tubular structure. The immunohistochemistry staining demonstrated keratin 18 and carcinoembryonic antigen were positively expressed in group C, which was similar to the tubular structure of secretory portion of sweat gland. Conclusion The sweat gland epithel ial cells can be induced to form eccrine sweat gland-l ike structure through three-dimensional culture in Matrigel.
Objective To study three-dimensional culturing methods of neonatal rat cardiac myocytes in simulated microgravity. Methods Neonatal rat primary cardiac myocytes were separated and seeded into polylactic acid scaffolds, stirredin spinner flasks for 24 hours, and then moved into rotary cell culture system for three-dimensional culture. The growth of cardiac myocytes was observed underinverted phase contrast microscope, scanning electron microscope and transmission electron microscope, and metabolic assay was assessed by MTT assay. Results Cardiac myocytes with sustained metabolic activity attached to the polylactic acid scaffolds, extended and confluenced. Pulsations of PLAcardiac myocytes was found in some areas. Conclusion The rotary cell culture system is suitable to develop neonatal rat cardiac myocytes culturing for three-dimensional modeling.
ObjectiveTo observe the growth characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) cultured on the alginate gel scaffolds and to explore the feasibility of hUCMSCs-alginate dressing for wound healing. MethodshUCMSCs were separated from human umbilical cords and cultured in vitro. After the 4th passage cells were co-cultured with alginate gel (experimental group), the cell growth characteristics were observed under the inverted phase contrast microscope. Vascular endothelial growth factor (VEGF) content was measured and the number of cells was counted at 0, 3, 6, and 9 days after culture; and the cell migration capacity was observed. The hUCMSCs were cultured without alginated gel as control. The model of full-thickness skin defects was established in 32 8-weekold Balb/c male mice and they were randomly divided into 4 groups (n=8): wounds were covered with hUCMSCsalginate gel compound (MSC-gel group), cell supernatants-alginate gel compound (CS-gel group), 10% FBS-alginate gel compound (FBS-gel group), and 0.01 mol/L PBS-alginate compound (PBS-gel group), respectively. Wound healing rates at 5, 10, and 15 days were observed and calculated; and the wound tissues were harvested for histological and immunohistochemical staining to assess new skin conditions at 15 days after operation. ResultshUCMSCs grew well with grape-like proliferation on the alginate gel, but no cell migration was observed at 7 days after cultivation. VEGF expression and cell number in experimental group were significantly less than those in control group at 3 days（P<0.05）; then they gradually increased, and VEGF expression and cell number were significantly more than those in control group at 9 days (P<0.05). The wound healing rates of MSC-gel and CS-gel groups were significantly higher than those of FBSgel and PBS-gel groups at 5, 10, and 15 days (P<0.05). The squamous epithelium, fibroblasts, sebaceous glands, capillaries and VEGF expression of the new skin in MSC-gel and CS-gel groups were significantly more than FBS-gel and PBS-gel groups (P<0.05). But there was no significance between MSC-gel and CS-gel groups (P>0.05). ConclusionhUCMSCs can continuously express VEGF in alginate gel, which is necessary for wound healing. The hUCMSCs-alginate compound is probably a good wound dressing.
ObjectiveTo study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. MethodsADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. ResultsThe result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P < 0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. ConclusionADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.
Objective To review the research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells (MSCs). Methods Recent literature about three-dimensional spheroid culture of MSCs was summarized, mainly on the formation of MSCs spheroids collected by three-dimensional culture, differences between MSCs spheroids and MSCs collected by traditional two-dimensional culture, and the mechanism underlying these differences. Last, its clinical prospect was discussed. Results Compared with MSCs collected by traditional two-dimensional culture, MSCs spheroids collected by three-dimensional culture get a salient up-regulation in anti-apoptosis, multiple differentiation potential, paracrine, and anti-inflammatory effect, which may be related to the morphology and cytoskeleton organization, cell-to-cell contact and gap junctions, and the hypoxia microenvironment. The animal experiments show obvious effects in repair of refractory wounds, repair of ischemic injury, and tissue remodeling, so MSCs spheroid has broad clinical prospect. Conclusion MSCs spheroids collected by three-dimensional culture have stronger biological potential and treatment effect than MSCs collected by traditional two-dimensional culture, MSCs spheroids can be used to optimize stem cell therapy and improve its treatment effect.