The concepts of “cytomedicine”, tissue engineering, and artificial organs have been developed in recent years, in which the heterotypic contact, cell-to-cell signaling, and uniform nutrient diffusion are key points. Artificial cells, called microencapsulated cells, can provide a liquid environment for cell cultures and maintain cell phenotype. They have potential for the immobilization of cells and enzymes, artificial organ translation, tissue engineering applications, etc. [1
] As presented in 1933, Bisceglie et al. sealed tumor cells in polymer membranes and transplanted them into the abdominal cavities of pigs. The results showed that the cells could survive long enough without being damaged by the immune system. Lim et al. [2
] combined microencapsulation techniques with tissue-cell transplantation to prepare sodium alginate/poly-lysine/sodium alginate (APA) microcapsules with good biocompatibility as an immune isolation tool. As reported in research, the “artificial cells” embedded with porcine islet cells successfully regulated blood glucose levels and replaced pancreatic function in rats, called the “artificial pancreas”. This research has opened up a new method for cell transplantation therapy.
Current limitations of artificial cells cultured in vitro are mass-transfer, cell viability, and phenotype stability [3
]. The microencapsulation of bioactive substances was first reported by Chang et al. in 1957. Microspheres prepared by hydrogel can provide a large surface area for cell growth and possess easy estimation for diffusion and mass-transfer behavior. Endres et al. [4
] reported that 3-(methacryloxy) propyl trimethoxysilane (MPS) stem cells implanted in microcapsules could express chondrocytes which were induced by TGF-β3. Moreover, Chan et al. [4
] prepared collagen microcapsules embedded in bone marrow mesenchymal stem cells and induced them to differentiate into chondrocytes, osteoblasts, and adipocytes in vitro. The results showed that cell encapsulation in hydrogels has been widely used in cytotherapy, regenerative medicine, 3D cell culture, and tissue engineering [6
]. The immunological isolation of artificial cells can effectively avoid immune rejection induced by homologous cells, xenogeneic cells, and genetically engineered cells after transplantation in vivo, while allowing sufficient diffusion of oxygen, nutrients, secreted molecules, and metabolic waste for the cells to survive and perform their functions [7
]. These results have broad application prospects in a variety of diseases and metabolic dysfunction treatments [8
It is necessary to strengthen the good biocompatibility of microcapsules to successfully apply microencapsulation technology to the medical field [9
]. Traditional construction has some disadvantages. For example, cells are sometimes difficult to grow into scaffolds and present uneven distribution. Different culture conditions can cause different viability in cells and further present different results in tissue engineering, which Liu has reported before [10
]. Compared to 2D conditions, 3D cultures can protect chondrocyte models from species-specific acute drug toxicity in vitro and can also maintain cell phenotype. To date, interest has focused on fundamental research into microspheres cultured in rotary cell culture system (RCCS) conditions and their application. Also, a very effective growth factor, basic fibroblast growth factor (bFGF), that induces the proliferation of chondrocytes, as well as angiogenesis and wound healing by affecting smooth muscle cells, endothelial cells, fibroblasts, and epithelial cells, has been broadly applied in tissue engineering [11
]. Studies have revealed that bFGF is beneficial for enhancing cell proliferation [12
] and retaining chondrocytes’ phenotype [13
The main components of the extracellular matrix are type II collagen, proteoglycans, glycosaminoglycans, hyaluronic acid, and some glycoproteins. The chondrocytes are terminally differentiated cells which are highly specific and their principal function is to maintain the stability of cartilage matrix components [14
]. Articular cartilage may be damaged due to illness, trauma, or aging bodies, which determines the limitation of self-repairing capability. The demand for cartilage cells is rapidly increasing for cell-based research and medicine [16
]. Herein, microencapsulated C5.18 cells were prepared by high-voltage electrostatic methods and the influences of different culture conditions with and without bFGF were investigated, which indicated a significant result in different culture conditions in vitro.
2. Materials and Methods
Sodium alginate was purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Cysteine, hematoxylin and eosin (H&E) staining kit, papain, and safranin-O were kindly purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Masson stain kit was obtained from Jiancheng Technology Co., Ltd. (Nanjing, China) C5.18 cells, acridine orange/ethidium bromide (AO/EB) and klcian blue staining Kit were provided by Syagen Biosciences Inc. (Guangzhou, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 1,9-dimethylmethylene blue (DMB) was obtained from Sigma Co., Ltd. (St. Louis, MO, USA). Rat collagen type-II (Col-II) ELISA kit was purchased from Cusabio Biotech Co., Ltd. (Wuhan, China). Basic fibroblast growth factor (bFGF) was purchased from PeproTech Inc. (Rocky Hill, CT, USA).
2.2. Preparation of Blank microcapsules and Microencapsulated C5.18 Cells (Artificial Cells)
As shown in Figure 1
, the blank microcapsules were prepared by using a WJN50 high-voltage electrostatic droplet generator (customized by Shanghai Ligong University) according to the work we reported elsewhere (parameters: 5.5 kV; 50 mm/h) [17
]. Briefly, cells in the logarithmic phase were digested with trypsin and the required cell concentration was adjusted to 5 × 106
cells/mL. The single cell suspension was firstly added into 2 mL of 1.5% alginate solution, which was further dropped into 1.5% CaCl2
solution by high-voltage electrostatic method (voltage: 5.5 kV; needle: 7#
; distance of needle and CaCl2
solution: 2 cm; propulsion speed: 50 mm/h) to obtain alginate-calcium (Alg-Ca) beads loaded with C5.18 cells. In order to form alginate/chitosan (AC) film, C5.18 cell-loaded Alg-Ca beads were made up to 0.5% chitosan solution (pH 5.5) for a 10-min film reaction after being filtered by tea leakage and the beads with the chitosan (CS) film were transferred to sodium alginate solution to form an AC film [10
]. Next, the liquefied core of the artificial cells was prepared with 1.6% sodium citrate solution for 2 min after being washed by saline. Those artificial C5.18 cells were finally diffused in complete medium in a culture flask with bFGF (100 ng/mL) under different conditions (static condition: 5% CO2
, 37 °C, saturated humidity; shaking conditions: 5% CO2
, 37 °C, saturated humidity, 60 r/min; rotary cell culture system: 5% CO2
, 37 °C, saturated humidity, 60 r/min). Control groups were managed with no bFGF addition.
2.3. Characterization of Morphology
Blank gel beads and cell-laden microcapsules were dispersed in PBS (pH 7.4) buffer. The morphology and variation of particle size could be observed by an optical microscope (Olympus Corporation, Tokyo, Japan). The particle size was determined by reading the size of 500 particles. The surface morphology of the gel beads was observed by a scanning electron microscope (Hitachi, Tokyo, Japan) after a lyophilized management.
2.4. Cell Proliferation Assay
CCK-8 assay was used to determine the proliferation activity of microencapsulated C5.18 cells. The cell-laden gels were seeded in 96-well plates with 100 μL completed medium per well, then cultured in a 5% CO2
incubator at 37 °C with saturated humidity. Each group had three parallel tests. The optical density at 450 nm (OD450
) value was directly in proportional to the number of C5.18 cells detected at vested culture time (1, 3, 5, 7, 10, 15, 20, and 30 days). 20 μL of 5 mg/mL CCK-8 was added into each well. After 4 h culturing, the 96-well plates were vibrated for 1 min using a microplate reader and the absorbance was measured at 450 nm using an enzyme-linked immunosorbent detector (Thermo, Waltham, MA, USA). The mean value was utilized to characterize the proliferation activity [18
2.5. AO/EB (Acridine Orange/Ethidium Bromide) Staining
200 μL of cell-laden droplet suspension was incubated with 10 μL of AO/EB solution for 30 s. 10 μL of suspension was placed onto a microscopic slide covering by a glass coverslip and then the sample was observed using a fluorescence microscope (Nikon TI-S, Japan) using a fluorescein filter and a 60× objective [19
2.6. Histological Staining
2.6.1. Hematoxylin-Eosin Staining of Cell Section
Microencapsulated cells sections were fixed in 4% paraformaldehyde for 30 min. Samples were washed by PBS 3 times and then placed onto a microscopic slide covered by a glass coverslip. Hematoxylin solution was dripped onto the slides for 12 min. The slides were rinsed in H2
O for 5 min and then were stained with 1% eosin Y solution for 5 min. The sections were dehydrated with two changes of 70% ethanol, two changes of 80% ethanol, and two changes of 100% ethanol for 30 sec each, then the ethanol was extracted with two changes of xylen for 5 min each. These slides were observed using confocal laser scanning microscopy (CLSM) (Leica company, Wetzlar, Germany) [20
2.6.2. Alcian Blue Stain
Microscope slides were incubated in 6-well plates with 400 μL laden-cells microcapsules suspension per well. After fixed in 4% paraformaldehyde for 30 min, the slides were stained with 1% alcian blue for 30 min. Later, the slides were respectively rinsed with PBS and absolute ethanol for 5 min and finally were observed using CLSM.
The hydrate slides were stained with safranin-O solution for 15 min and then washed in running tap water for 5 min. The slides were dehydrated with 95%, and100% alcohol for 20 sec each. Next, the slides were rinsed in xylen for 10 min. Samples were eventually observed under CLSM after being mounted in mounting medium.
2.7. Immunohistochemistry (IHC) Staining
Protocols for immunohistochemistry staining briefly were as follows: Each fixed section was placed in 0.5% triton X-100 solution for 20 min. After being fixed with 4% paraformaldehyde, the slides with cells were immersed in 0.5% triton X-100 for 20 min, then washed in PBS 3 times for 20 min each. After being incubated in 3% H2O2 for 15 min, the slides were washed with PBS 3 times for 2 min each and then were blocked in buffer containing 0.5% BSA and 2% FBS for 15 min. After that, the slides were placed in the diluted primary antibody (collagen II antibody) for 60 min at 37 °C, then were incubated with secondary antibody (horseradish peroxidase labeled goat anti-rabbit IgG) in a humidified chamber at room temperature. 20 min later, samples were rinsed with PBS 5 times for 2 min each and stained with 3,3′-diaminobenzidine (DAB) and hematoxylin for 10 min, respectively. Finally, the slides were washed in running water for later detection.
2.8. Glycosaminoglycan (GAG) Release 
Quantification of total glycosaminoglycans (GAG) in chondrocyte cultures is necessary for the complete assessment of the metabolic profile of the system and is commonly performed using the 1,9-dimethylmethylene blue (DMB) dye method [22
]. The artificial cells obtained after determinate time were cultured with 300 μL papain in a water-bath at 60 °C overnight and then were centrifuged at 4000 rpm for 5 min to obtain supernatant samples. 100 μL of papain-digested samples were added into 2.5 mL DMB solution. Absorbance was determined at 525 nm. GAG content of the samples was determined using a C-6-S standard curve (0–100 μg/mL).
2.9. Type II Collagen Secretion
Artificial cells were washed by PBS. After being broken, suspension was centrifuged at 5000 rpm for 5 min to obtain subsidence-containing cells. Next, 1 mL of PBS was added and samples were stored at −20 °C overnight. After being centrifuged at 5000 rpm for 5 min, the obtained supernatant was detected at 450 nm using the type II collagen ELISA kit assay.
2.10. Statistical Analysis
Results were expressed as mean ± standard deviation. Statistical significance was measured by one-way ANOVA. * p < 0.05 and ** p < 0.01 were considered significant. Each measurement reported was based on duplicate analysis of at least three independent experiments.
Artificial cells were prepared using a high-voltage electrostatic method through microcapsule-loaded C5.18 chondrocytes using alginate and chitosan. Under optimized conditions, it obtained microencapsulated cells with uniform cell distribution, good sphericity, and a smooth surface by embedding a different cell density. The particle size distribution was 150–280 μm with an average particle diameter of 220 μm. AO/EB and H&E staining indicated high cell viability and the typical morphology of chondrocytes (time). The content of GAG and Col-II detection showed that the constructed C5.18 cells under 3D microgravity conditions could improve cells to form large cell aggregates with bFGF joining, which provides the possibility for cartilage tissue constructs in vitro.
Nutrients were placed under static environments and the presence of the chitosan membrane in microcapsules hindered communication between the cells and culture medium [29
]. Furthermore, metabolic waste was present in the system at all times [30
]. As a result, cells began to apoptose and die. Without the presence of bFGF, nutrition could get into microcapsules and constructs under dynamic conditions. Thus, cells under shaking and RCCS conditions survived longer than those under static conditions. However, the presence of chitosan still limited the contact between the cells and the culture medium. The presence of shear forces produced under dynamic conditions hindered cell growth, thus some cells started apoptosis. When bFGF was added, the values of the cells increased significantly, as bFGF could promote cell mitosis and therefore, cell proliferation. Cell mitosis and cellular uptake of nutrients could be promoted by bFGF and dynamic conditions, respectively, so the number of cells increased with increasing GAG and Col-II synthesis. Altogether, the cell viability and the corresponding amount of GAG and Col-II of chondrocytes were improved.
In summary, the results demonstrated that the microencapsulated C5.18 cells incubated in the medium containing bFGF under RCCS conditions could increase the number of cells with a chondrogenic phenotype, which further presents a method to obtain meaningful numbers of cells in artificial cells using the microencapsulation technique and provides a promising pathway to cartilage regeneration in vitro.