As a high-quality moxibustion material, Artemisia stolonifera has high economic value and research prospects. However, due to difficulties in seed germination, its wild germplasm resources are sparsely distributed in China. This study used young stem segments grown in the current year to investigate the effects of explant sterilization, different combinations and concentrations of plant growth regulators on the proliferation and rooting of adventitious shoots, with the aim of constructing an in vitro rapid propagation technology system for A. stolonifera. The results showed that the lowest contamination rate of 25.83% was achieved when sterilizing the stem segments by rinsing with running water for 30 min, soaking in 75% ethanol for 30 s, followed by a 5 min treatment with 0.1% HgCl_2, 10 min with 8% NaClO, and 10 min with 0.6% phytosaniline. There was no browning of the stem segments, and surface sterilization of the A. stolonifera stem segments was successfully achieved. In the induction culture phase, when the concentration of kinetin(KT) was 0.05 mg·L~(-1) and 6-benzylaminopurine(6-BA) was 0.05 mg·L~(-1), the adventitious shoot proliferation coefficient was 2.02, effectively promoting the proliferation and growth of A. stolonifera. In the rooting culture phase, 0.1 mg·L~(-1) 1-naphthaleneacetic acid(NAA) effectively induced A. stolonifera test-tube seedlings to root within a short period, achieving a rooting rate of 100%. The addition of a small amount of activated charcoal also promoted rooting and strengthened seedling growth. The survival rate of A. stolonifera seedlings transplanted into a substrate consisting of 90% nutrient soil and 10% perlite was 100%. This study established an efficient in vitro rapid propagation system for A. stolonifera, overcoming difficulties with seed germination, shortening the breeding cycle, and reducing production and planting costs. It provides technical support for the introduction, domestication, seedling propagation, germplasm conservation, and industrial development of A. stolonifera.
Artemisia/drug effects* ; Tissue Culture Techniques/methods* ; Plant Growth Regulators/pharmacology* ; Plant Stems/drug effects* ; Plant Shoots/drug effects*

Organoids are three-dimensional (3D) cellular structures formed through the differentiation and self-organization of pluripotent stem cells or tissue-derived cells, showing considerable potential in the research on disease mechanism, personalized medicine, and developmental biology. However, the development of organoids is limited by the complex composition, batch-to-batch variations, and immunogenicity of basement-membrane matrix in the current culture system, which hinders the clinical translation and in vivo applications of organoids. Hydrogels are highly hydrated 3D polymer network materials, with modifiable mechanical and biochemical properties by engineering, representing an ideal alternative to basement-membrane matrix. This article reviews the research progress in engineered hydrogels with defined composition currently used in organoid culture. We introduce the structural characteristics and engineering design considerations of hydrogels, emphasize the latest research progress and specific application cases, and discuss the future development of these engineered hydrogels, provide valuable insights for the further advancement and optimization of engineered hydrogels for organoid.
Hydrogels/chemistry* ; Organoids/cytology* ; Tissue Engineering/methods* ; Humans ; Animals ; Pluripotent Stem Cells/cytology* ; Cell Culture Techniques, Three Dimensional/methods* ; Tissue Scaffolds

OBJECTIVE: To review the application of cell derived decellularized extracellular matrix (CDM) in tissue engineering. METHODS: The literature related to the application of CDM in tissue engineering was extensively reviewed and analyzed. RESULTS: CDM is a mixture of cells and their secretory products obtained by culturing cells in vitro for a period of time, and then the mixture is treated by decellularization. Compared with tissue derived decellularized extracellular matrix (TDM), CDM can screen and utilize pathogen-free autologous cells, effectively avoiding the possible shortcomings of TDM, such as immune response and limited sources. In addition, by selecting the cell source, controlling the culture conditions, and selecting the template scaffold, the composition, structure, and mechanical properties of the scaffold can be controlled to obtain the desired scaffold. CDM retains the components and microstructure of extracellular matrix and has excellent biological functions, so it has become the focus of tissue engineering scaffolds. CONCLUSION CDM is superior in the field of tissue engineering because of its outstanding adjustability, safety, and high bioactivity. With the continuous progress of technology, CDM stents suitable for clinical use are expected to continue to emerge.
Tissue Engineering/methods* ; Tissue Scaffolds/chemistry* ; Humans ; Decellularized Extracellular Matrix/chemistry* ; Cells, Cultured ; Extracellular Matrix ; Animals ; Biocompatible Materials/chemistry* ; Cell Culture Techniques

In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.
Animals ; Organoids/metabolism* ; Intestine, Small/drug effects* ; Chickens ; Cell Culture Techniques/methods* ; Culture Media ; Chick Embryo ; Tissue Culture Techniques/methods*

Diabetes constitutes a worldwide epidemic that affects all ethnic groups. Cell therapy is one of the best alternatives of treatment, by providing an effective way to regenerate insulin-producing cells lost during the course of the disease, but many issues remain to be solved. Several groups have been working in the development of a protocol capable of differentiating Mesenchymal Stem Cells (MSCs) into physiologically sound Insulin Producing Cells (IPCs). In order to obtain a simple, fast and direct method, we propose in this manuscript the induction of MSCs to express NESTIN in a short time period (2 h), proceeded by incubation in a low glucose induced medium (24 h) and lastly by incubation in a high glucose medium. Samples from cell cultures incubated in high glucose medium from 12 to 168 h were obtained to detect the expression of INSULIN-1, INSULIN -2, PDX-1 and GLUT-2 genes. Induced cells were exposed to a glucose challenge, in order to assess the production of insulin. This method allowed us to obtain cells expressing PDX-1, which resembles a progenitor insulin-producing cell.
Cell Culture Techniques ; Cell- and Tissue-Based Therapy ; Ethnic Groups ; Glucose ; Humans ; Insulin* ; Mesenchymal Stromal Cells* ; Methods ; Nestin*

PURPOSE: The purpose of this study was to investigate the effects of transplantation of an in vitro-generated, scaffold-free, tissue-engineered cartilage tissue analogue (CTA) using a suspension chondrocyte culture in a rabbit growth-arrest model. MATERIALS AND METHODS: We harvested cartilage cells from the articular cartilage of the joints of white rabbits and made a CTA using a suspension culture of 2x107 cells/mL. An animal growth plate defect model was made on the medial side of the proximal tibial growth plate of both tibias of 6-week-old New Zealand white rabbits (n=10). The allogenic CTA was then transplanted onto the right proximal tibial defect. As a control, no implantation was performed on the left-side defect. Plain radiographs and the medial proximal tibial angle were obtained at 1-week intervals for evaluation of bone bridge formation and the degree of angular deformity until postoperative week 6. We performed a histological evaluation using hematoxylin-eosin and Alcian blue staining at postoperative weeks 4 and 6. RESULTS: Radiologic study revealed a median medial proximal tibial angle of 59.0degrees in the control group and 80.0degrees in the CTA group at 6 weeks. In the control group, statistically significant angular deformities were seen 3 weeks after transplantation (p<0.05). On histological examination, the transplanted CTA was maintained in the CTA group at 4 and 6 weeks postoperative. Bone bridge formation was observed in the control group. CONCLUSION: In this study, CTA transplantation minimized deformity in the rabbit growth plate injury model, probably via the attenuation of bone bridge formation.
Animals ; *Bone Transplantation ; Cartilage/anatomy & histology ; Cell Culture Techniques ; Cells, Cultured ; Chondrocytes/*cytology/transplantation ; Growth Plate/anatomy & histology/*surgery ; *Mesenchymal Stem Cell Transplantation ; Rabbits ; Tibia/*surgery ; Tissue Engineering ; Transplantation, Autologous/methods ; Transplantation, Homologous

In tissue engineering, alginate has been an attractive material due to its biocompatibility and ability to form hydrogels, unless its uncontrollable degradation could be an undesirable feature. Here, we developed a simple and easy method to tune the degradation profile of the fibrous alginate scaffolds by the microfluidic wet spinning techniques, according with the use of isopropyl alcohol for dense packing of alginate chains in the microfiber production and the increase of crosslinking with Ca²⁺ ion. The degradation profiling was analyzed by mass losses, swelling ratios, and also observation of the morphologic changes. The results demonstrated that high packing density might be provided by self-aggregation of polymer chains through high dipole interactions between sheath and core fluids and that the increase of crosslinking rates could make degradation of alginate scaffold controllable. We suggest that the tunable degradation of the alginate fibrous scaffolds may expand its utilities for biomedical applications such as drug delivery, in vitro cell culture, wound healing, tissue engineering and regenerative medicine.
2-Propanol ; Cell Culture Techniques ; Hydrogel ; Hydrogels ; In Vitro Techniques ; Methods ; Microfluidics* ; Polymers ; Regenerative Medicine ; Tissue Engineering ; Wound Healing

Breeding is not only an important area of medicinal plants research but also the foundation for the superior varieties acquirement of medicinal plants. The rise of modern biotechnology provides good opportunities and new means for medicinal plants breeding research in China. Biotechnology shows its technical advantages and new development prospects in breeding of new medicinal plants varieties with high and stable yield, good quality, as well as stress-resistance. In this paper, we describe recent advances, problems, and development prospects about the application of modern biotechnology in medicinal plants breeding research in China.
Biotechnology ; methods ; Breeding ; China ; Plants, Medicinal ; genetics ; Research ; Tissue Culture Techniques

Using MS as basic medium, supplemented with 1.0 mg · L(-1) IBA, the adventitious roots of Tripterygium wilfordii were induced, and the good adventitious root culture system was established by leaves or callus induced by leaves as explants. The adventitious roots were also induced with 2.0-4.0 mg · L(-1) NAA and the good adventitious root culture system established by using suspension cells from callus as materials to induce adventitious root. The content of triptolide of three adventitious roots culture system were exceeded in the natural root bark. The content of triptolide of AR3 adventitious roots was the highest about 5.3 times as that in the natural root bark. By using 5 L stirred fermentor during pilot enlarge cultivation, compared with 250 mL flask cultivation, the adventitious roots increment and secondary metabolites content per liter medium showed no significant difference. The accomplishment of this analysis laid a foundation by tissue culture production of the secondary metabolites of T. wilfordii.
Culture Media ; chemistry ; metabolism ; Fermentation ; Plant Growth Regulators ; analysis ; metabolism ; Plant Roots ; growth & development ; metabolism ; Tissue Culture Techniques ; instrumentation ; methods ; Tripterygium ; growth & development ; metabolism

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis