Construction strategies of skin organoids: a review / 中华创伤杂志

Large skin defect caused by severe trauma is a common clinical problem with high incidence, great harm, difficult treatment and poor prognosis, which not only seriously affects the quality of patients′ life, but also threatens their lives. Large skin defects are difficult to heal by themselves and the main treatment is skin transplantation. However, the source of the autologous flap is limited and may cause secondary damage to patients. The artificial skin has poor mechanical integrity that cannot be integrated, causing formation of scars, and also has the risk of immune rejection. Skin organoid technology can extremely simulate the human skin tissue and its functions. Thus, it can overcome the shortcomings of the current skin wound treatment to a certain extent and provide a new treatment for the patients with large skin defects. At present, the construction methods of skin organoids are relatively mature, but each method has its advantages and disadvantages, and the best method has not been determined yet. Moreover, the structure and function of skin organoids are relatively simple, so there is still a relatively big gap between skin organoids and real human skin. Hence, the authors reviewed the research progress in skin organoid construction strategies from organoids′ skin organoid technology, and construction methods of skin organoids, hoping to provide a reference for the construction of skin organoids with more complex structures and functions in the future.

Cell-loaded hydrogel microspheres based on droplet microfluidics: a review / 生物工程学报

Droplet microfluidics technology offers refined control over the flows of multiple fluids in micro/nano-scale, enabling fabrication of micro/nano-droplets with precisely adjustable structures and compositions in a high-throughput manner. With the combination of proper hydrogel materials and preparation methods, single or multiple cells can be efficiently encapsulated into hydrogels to produce cell-loaded hydrogel microspheres. The cell-loaded hydrogel microspheres can provide a three-dimensional, relatively independent and controllable microenvironment for cell proliferation and differentiation, which is of great value for three-dimensional cell culture, tissue engineering and regenerative medicine, stem cell research, single cell study and many other biological science fields. In this review, the preparation methods of cell-loaded hydrogel microspheres based on droplet microfluidics and its applications in biomedical field are summarized and future prospects are proposed.

Progress on three-dimensional cell culture technology and their application / 生物医学工程学杂志

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3 µm, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14 µm. The fibers were connected by pores, and the pore diameter was (65±35) µm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P < 0.01). (2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P < 0.01). (4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

Effects of TGF-β1 on the migration of oral cancer-associated fibroblasts in two and three dimensional co-culture models / 口腔疾病防治

Objective@#To observe the effect of transforming growth factor-β1 (TGF-β1) on the migration of oral carcinoma associated fibroblasts (CAFs) with two-dimensional culture model and three-dimensional model.@*Methods @# Under two-dimensional culture conditions, CAFs stimulated by TGF-β1 with the addition of 10 ng/mL medium were used as the experimental group, and untreated CAFs were used as the control group. The migration of CAFs with the stimulation of TGF-β1 was measured by cell scratch assay and transwell assay. CAFs positive for green fluorescent protein (GFP) were cultured by retrovirus transfection. Human tongue squamous cell carcinoma cells SCC25, GFP(+) CAFs and CAFs with three-dimensional cell co-culture models were established. The three-dimensional model cultured under the stimulation of TGF-β1 with 10 ng/mL medium was used as the experimental group, and the three-dimensional model without TGF-β1 was used as the control group. The migration of CAFs with the stimulation of TGF-β1 was also measured by the three-dimensional models.@*Results@# It was verified that 10 ng/mL TGF-β1 promoted the migration of CAFs in the two-dimensional culture model. The three-dimensional co-culture models of SCC25, GFP(+) CAFs and CAFs were successfully established. The migration of SCC25 and CAFs was detected in the three-dimensional model. However, 10 ng/mL TGF-β1 had little effect on their migration.@*Conclusion@#The effect of TGF-β1 in vitro on the migration of oral CAFs was associated with different culture models in two and three dimensions.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3μ m, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14μm. The fibers were connected by pores, and the pore diameter was (65±35) μm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P<0.01).(2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P<0.01).(4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

Progress of multi-cellular tumor spheroids culture technique for treatment of bladder cancer / 肿瘤研究与临床

Cell culture is an important tool for biological research.To better understand the pathogenesis and therapeutic methods of the tumors,the three-dimensional cell culture is applied by more and more researchers to create a culture environment that closes to the tumor microoenvironment.Thanks to the advances in the tissue engineering technology,many kinds of models of the three-dimensional cell culture achieve wide accessibility.Compared with the traditional two-dimensional cell culture,the three-dimensional cell culture is better in simulating physiological features of the human histology and cells,including cell proliferation and differentiation,the interaction of cell to cell and cell to matrix.This paper reviews the progress of multi-cellular tumor spheroids (MCTS) culture technique of the three-dimensional cell culture for treatment of bladder cancer.

Microfluidic chip for tumor cell 3 D culturing establishment and its cultural conditions / 中国药理学通报

Aims To design and fabricate the 3 D cell cultural microfluidic chip for tumor cell culturing, with which to research the compatible conditions for gelatin forming and dissolving with calcium alginate as the scaffolds. To culture SMMC-7721 cells in the chip and to detect the surviving rate. Methods The microfluid-ic chip was fabricated with the software Corel Draw, the technology of soft lithography, molding, and plas-ma bonding. The applicability was tested and cells were cultured on it, on which the cell status was ob-served, their surviving rate was calculated with the help of the software IPP. Results The chip we fabri-cate was calculated is suitable for cell 3D culturing, the tumor cells showed a favorable proliferation ability in 72 h on chip, the surviving rate was ( 96. 1 ± 4. 5)%. The cells were solid and TCS appeared. Con-clusion The microfluidic chip manufactured appeared for tumor cell 3D culture, is suitable for the growth of SMMC-7721 and the cells are indubitable. They show some different status in proliferative and agminated compared with traditional 2D cell culture. With the chip and the condition found, there will be a better way to study the characteristics of tumor cells and is beneficial to the screen of anti-tumor drugs.

Influence of different culture methods in DNA methylation status of breast cancer MDA-MB-23 1 cells / 吉林大学学报(医学版)

Objective:To explore the influence of different cell culture methods in the genome-wide DNA methylation status of breast cancer MDA-MB-231 cells,and to clarify the relationship between genome-wide DNA methylation status and cell growth environment and the role of genome-wide DNA methylation status in the occurrence and development of tumor.Methods:The MDA-MB-231 cells were cultured with 2D and 3D cell culture models and mouse orthotopic transplantation model (Ti model)and collected, then DNA was extracted by DNA extraction kit and the genome-wide DNA methylation status of MDA-MB-231 cells after cultured with three different culture methods was detected by DNA methylation chip,then the value of beta,DiffScore and Delta_Beta of the CpG loci of each gene were calculated by applying Genomestudio software, and the differential methylation genes were screened by Genomestudio software and GO and Pathway analysis of these genes were performed in DAVID on-line analysis tool.Results:All 480 genes of the MDA-MB-231 cells showed significant differences in the degree of methylation in 3D and 2D models (P<0.05);86 448 genes in 3D and Ti models (P<0.05);90 005 genes in Ti and 2D models (P<0.05).The differential methylation genes in 3D and 2D,3D and Ti,and Ti and 2D models were enriched on the multicellular organismal development term and cell differentiation term (P<0.05);also on MAPK signaling pathway,cell adhesion molecules (CAMs),and regulation of actin cytoskeleton (P<0.05). Conclusion:There are differences in genome-wide DNA methylation status of MDA-MB-231 cells cultured in 2D, 3D cell culture and Ti models.

Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Adipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition.</p><p><b>METHODS</b>ASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture.</p><p><b>RESULTS</b>The 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs' potential of cellular transformation was lower after irradiation by soft agar assay.</p><p><b>CONCLUSION</b>These findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.</p>

Functionalized polypeptide hydrogel matrix for ex vivo modulation of proliferation and differentiation of neural stem cells / 中华神经医学杂志

Objective In vitro model with three dimensional cell culture provides the appealing biomimetic platform to probe the biological characteristics of multiple stem cells,which serves as an important in vitro tools to investigate regulating factors controlling the proliferation and differentiation of neural stem cells (NSCs).The present study aims to reconstruct an integrated poly-peptide hydrogel made extracellular matrix (ECM) enhanced with cyclo-RGD molecular [c(RGDfK)] for the exploration of NSCs bio-characteristics.Methods Spinal cords from one-d-old SD rats were collected and spinal-derived NSCs were induced in the complete medium; immunofluorescence staining was employed to observe the NSCs morphology and identify NSCs.Three hydrogel including type Ⅰ collagen,self-assembly poly-peptide nanofiber hydrogel (SAPNH) of RADA16 and RADA16-c(RGDfK) were employed to serve as culturing ECM of spinal-derived NSCs to mimic the ex vivo 3-D culturing.With the theological analysis,cyto-morphological observation was performed,NSCs proliferation was observed by MTT assay,and cell immunochemistty and confocal microscopy were employed to detect the NSCs differentiation.Results SAPNH born appropriate elastic module conducive to the cellular adhesion and proliferation of neural cells (RADA16 and RADA16-c (RGDfK)=(0.42±0.07) kPa and (0.47 ±0.09) kPa,without significant difference (P＞0.05); however,the elastic module in the type Ⅰ collagen group was (0.87±0.12) kPa,which was significantly stronger than the two groups (P＜0.05).Uniform distribution of neuron-shape cells was noted in the extracellular matrix ofRADA16-c (RGDfK) cells,with almost the diameter of cell sphere (200-300 μm); cells in the RADA16 and RADA16-c (RGDfK) had better growth characteristics than the other two groups; RADA16-c(RGDfK) had significantly better cellular adhesion and proliferation of neural cells as compared with RADA16 and collagen groups (P＜0.05).A large number of neural precursor cells differentiated into neurogliocytes was noted in the RADA16 group and RADA16-c (RGDfK) group,with low proportion of neuronal differentiation (17.6％ ±3.1％ and 19.0％±3.6％,respectively); this proportion of neuronal differentiation was significantly higher than that in the type Ⅰ collagen group (10.6％±2.3％,P＜0.05).Conclusions The functionalized SAPNH enhanced with c(RGDfK) presents the excellent biocompatibility and promotes the adhesion and proliferation of spinal NSCs.Serving as the engineered cellular vector,functionalized SAPNH has laid a solid foundation for the studies of neural regeneration and repair with novel neuro-engineering techniques in the subsequent researches.

Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro / 华中科技大学学报（医学）（英德文版）

o-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

The application of three-dimensional cell culture technology in the field of cancer research / 中国药理学通报

Traditional 2D cell culture technology is widely used in cell culture field, but it lacks the formation of 3D matrix scaffold and lacks specific cytokines in vivo.Cells in 3D cell culture system are similar to cells in vivo for gene expression and cell activity. The article focuses on the application of three-dimensional cell culture technology in cancer research, including the construction of tumor microenvironment,tumor biological behavior, tumor angiogenesis, drug resistance and so on and provides a reference for cancer research workers.

The changes in biological characteristics of lung adenocarcinoma A549 cells under two in vitro culture systems / 解放军医学杂志

Objective To investigate the changes in cells on cell cycle,cell apoptosis and susceptibility to chemotherapeutic drugs of lung adenocarcinoma A549 cells under two dimensional (2D system) and three dimensional (3D system) culture conditions. Methods The three dimensional culture of A549 cells was carried out using rotatable culture bottle and desk-top water bath shake. Flow cytometer,in situ cell apoptosis detection kit,MTT and blood cell counter were employed to compare cell cycle,cell apoptosis and susceptibility to ADM of A549 cells under different culture conditions. Results The three dimensional culture system was confirmed to have been successful by the aid of observation under inverted and electron microscope. There were significant retardation of G_ 1 phase,lower cell apoptosis rate and decreased susceptibility to ADM in A549 cells in 3D system compared with those in 2D system. Conclusions There were remarkable differences in biological characteristics of A549 cells in two culture systems,indicating that A549 cells cultivated in 3D system simulated better solid tumor in vivo . The 3D system was very useful for further investigation of the behavior of solid tumor,so that anti-carcinoma chemotherapeutic drugs could be advantageously tested in vitro before clinical application.