Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

OBJECTIVETo acquire lots of cell to culture during the primary cell culture.

METHODWe take the split-thickness skin slide technique to acquire the dissociated fibroblast cell in two big-ear rats.

RESULTSThe cell number is above 10(6) from 1 cm x 2 cm split-thickness skin slide and the technique is simple, economic, effectve.

CONCLUSIONWe think this way is better than other methods, and should be adopted in the primary cell culture, especially in fibroblast transplantation by injection.

Animals ; Cell Culture Techniques ; methods ; Fibroblasts ; cytology ; transplantation ; Rabbits

A line of fibroblast-type cells derived from embryos of a domestic rabbit has been cultivated continuously for over 3 years by serial passages up to the level of the moth passage. The cell line was tentatively named rabbit embryo fibroblast (REF). The establishment of primary culture, serial passages, growth rate and cytology are described in this communication. In addition some of the results of experiments on the detection of Mycoplasma contamination, on storage of the frozen cells and on its susceptibility to vaccinia virus infection are included.
Animal ; Cell Line ; Embryo/cytology* ; Fibroblasts* ; Rabbits ; Tissue Culture*

OBJECTIVETo explore the way of stably inducing canine bone marrow mesenchymal stem cells (BMSCs) to differentiate into fibroblasts and myofibroblasts in vitro, and provide seed cells for fabricating tissue engineering heart valves (TEHV).

METHODSAdult canine BMSCs were separated by a gradient centrifugation on Percoll (density 1.073 g/ml), then the cells were incubated in low-glucose Dulbecco Eagle's minimum essential medium (LG-DMEM) with 10% bovine calf serum. Cell phenotype were identified by immunohistochemistry staining. The second and third generation of BMSCs were committedly induced by conditioning culture medium, which were detected by immunohistochemistry staining. The induced-BMSCs were freezed, preserved and resuscitated after 7 d to observe the cell growth, proliferation and function.

RESULTSBMSCs deriving from the bone marrow mononuclear cells separated by a Percoll gradient were positive expression of alpha-smooth muscle antibody, vimentin and negative expression of CD34, laminin. About (50 +/- 3)% induced-BMSCs were positive expression of laminin. Approximately (85 +/- 3)% freezed induced-BMSCs could be resuscitated. And the growth, proliferation and function were well.

CONCLUSIONBMSCs could be committedly induced to differentiate into fibroblasts and myofibroblasts in vitro. It is suitable to be the seed cells.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Dogs ; Fibroblasts ; cytology ; Mesenchymal Stromal Cells ; cytology ; Monocytes ; cytology ; Myoblasts ; cytology

OBJECTIVETo explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro.

METHODSPorcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results.

RESULTSIn chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology.

CONCLUSIONThe 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.

Animals ; Cartilage ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Dermis ; cytology ; Fibroblasts ; cytology ; Humans ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs).

METHODSSSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR.

RESULTSSSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes.

CONCLUSIONThe feeder layer of hEFs supports the growth of human spermatogonial stem cells.

Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; Humans ; Male ; Spermatogonia ; cytology ; Stem Cells ; cytology

The proliferation of cardiac fibroblasts (CFs) is a key pathological process in the cardiac remodeling. To establish an objective, quantitative method for the analysis of cell proliferation and cell cycle, we applied the high-content screening (HCS) and flow cytometry (FCM) techniques. CFs, isolated by enzyme digestion from newborn C57BL/6J mice, were serum starved for 12 h and then given 10% fetal bovine serum (FBS) for 24 h. Followed by BrdU and DAPI (or 7-AAD) staining, CFs proliferation and cell cycle were analyzed by HCS and FCM, respectively. Discoidin domain receptor 2 (DDR2) staining indicated that the purity of isolated CFs was over 95%. (1) HCS analysis showed that the ratio of BrdU-positive cells was significantly increased in 10% FBS treated group compared with that in serum-free control group [(12.96 ± 0.67)% vs (2.77 ± 0.33)%; P < 0.05]. Cell cycle analysis showed that CFs in G0/G1 phase were diploid, and CFs in S phase were companied with proliferation, DNA replication and enlarged nuclei; CFs in G2 phase were tetraploid, and CFs in M phase produced two identical cells (2N). (2) FCM analysis showed that the ratio of BrdU-positive cells was increased in 10% FBS treated group compared with that in the control group [(11.10 ± 0.42)% vs (2.22 ± 0.31)%; P < 0.05]; DNA content histogram of cell cycle analysis indicated that the platform of S phase elevated in 10% FBS group compared with control group. (3) There were no differences between the two methods in the results of proliferation and cell cycle analysis. In conclusion, HCS and FCM methods are reliable, stable and consistent in assessment of the proliferation and cell cycle in CFs.
Animals ; Cell Cycle ; Cell Proliferation ; Fibroblasts ; cytology ; Flow Cytometry ; Mice ; Mice, Inbred C57BL ; Mitosis ; Myocardium ; cytology

Scar, either hypertrophic scar or keloid, is one of the most common complications due to proliferative disorder of fibrosis in the process of wound healing after burn injuries, trauma, and surgical operations. To repair the cosmetic and functional impairments caused by scars poses a great challenge to all the burn surgery workers. With the advances in both basic research and clinical treatment, the understanding of scar formation and the therapeutic strategies of scar have been improved significantly. However, the remaining problems are still outstanding. In this discussion, the advances and problems in the scientific research in this field, including genetic predisposition, candidate gene, dysfunction of fibroblasts, interaction between fibroblasts and keratinocytes, as well as animal models for hypertrophic scar and keloid were summarized. In addition, the progresses in the clinical therapies are also discussed, including pressure treatment, silicone gel sheeting, corticosteroids, laser, and other emerging treatment strategies. The understanding and treatment of scar will improve in the future with further deepening basic research and clinical trials with stricter standard of assessment.
Burns ; pathology ; Cicatrix ; genetics ; pathology ; therapy ; Fibroblasts ; cytology ; Humans ; Keratinocytes ; cytology

OBJECTIVETo investigate the feasibility of construction of tissue-engineered skin in vitro.

METHODSFibroblasts were seeded on the dermal surface of acellular dermal matrix (ADM). 7 days later, epidermal cells (5 x 10(5)/cm2) were mixed with fibroblasts (0.2 x 10(5)/cm2) and then seeded on epidermal surface of ADM. The culture medium was the mixture liquor containing K-SFM in half and the culture supernatants of fibroblasts in half. In the control group, only epidermal cells (5 x 10(5)/cm2) were seeded and cultured with K-SFM. After composite skin was cultured for 1 week and 3 week, samples were harvested respectively for morphological study and to receive identification by immunohistochemistry.

RESULTSAfter 3-week culture, there were 3-4 continuous layers of cells in the epidermis in the experiment group. The epidermis was attached tightly to the dermis with trochanterellus. But in the control group, there were just 1-2 layers of cells in the epidermis which was not connected to the dermis. Strong positive dye of Laminin indicated that basement membrane was thoroughly formed in the experiment group. The same result was demonstrated by transmission electron microscope.

CONCLUSIONSThe mix-seeding of epidermal cells and few fibroblasts would promote the adhesion and proliferation of epidermal cells on ADM, and the formation of basement membrane.

Animals ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; Fibroblasts ; cytology ; Skin, Artificial ; Swine ; Tissue Engineering ; Tissue Scaffolds

Bone Marrow Cells ; cytology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; Humans ; Myocardium ; pathology