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1.
Cryo Letters ; 27(6): 361-8, 2006.
Article in English | MEDLINE | ID: mdl-17256070

ABSTRACT

Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0 degree C to -35 degree C at a cooling rate of 0.5 degree per min, with seeding was set at -10 degree C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of 11 colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.


Subject(s)
Cryopreservation/methods , Embryonic Stem Cells/physiology , Algorithms , Cell Differentiation/physiology , Cell Survival/physiology , Cells, Cultured , Cold Temperature , Humans , Time Factors
2.
Shi Yan Sheng Wu Xue Bao ; 38(3): 247-56, 2005 Jun.
Article in Chinese | MEDLINE | ID: mdl-16044919

ABSTRACT

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler was used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rates, the temperature of seeding, the temperatures before the samples being plunged into liquid nitrogen, and the cryoprotective agents. It was found that the favorable constitution of cryoprotective agents was Me2SO+ FBS+DMEM(1:3:6, v/v/v) with cooling protocol of -0.5 degrees C/min from 0 degrees C to -35 degrees C (seeding at -10 degrees C), and being plunged into the liquid nitrogen immediately. The high survival rate (81.8%) was obtained.


Subject(s)
Cryopreservation/methods , Embryonic Stem Cells , Cell Line , Humans
3.
Reprod Biomed Online ; 11(6): 733-9, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16417738

ABSTRACT

Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1-60, TRA-1-81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells.


Subject(s)
Cryopreservation/methods , Embryo, Mammalian/cytology , Pluripotent Stem Cells , Trehalose , Animals , Base Sequence , Cell Differentiation , Cell Line , Cryoprotective Agents , DNA, Complementary/genetics , Female , Humans , In Vitro Techniques , Male , Mice , Mice, SCID , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation , Transplantation, Heterologous
4.
Cell Res ; 13(5): 335-41, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14672556

ABSTRACT

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 106 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.


Subject(s)
Blood Vessel Prosthesis , Cell Differentiation/physiology , Endothelium, Vascular/cytology , Stem Cells/cytology , Tissue Engineering/methods , Animals , Cell Line , Endothelium, Vascular/embryology , Mice , Mice, Nude , Muscle, Smooth, Vascular/cytology , Rabbits
5.
Cell Res ; 13(3): 195-202, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12862320

ABSTRACT

As a part of a basic research project on Xeno-transplantion, we have been engaged in the derivation of embryonic stem cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 and a 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentially useful for further basic studies.


Subject(s)
Cell Culture Techniques/methods , Embryo, Mammalian/cytology , Germ Cells/cytology , Transcription Factors , Alkaline Phosphatase/analysis , Animals , Antigens, Surface/immunology , Biomarkers/analysis , Cell Differentiation/physiology , DNA-Binding Proteins/immunology , Embryo, Mammalian/chemistry , Embryo, Mammalian/ultrastructure , Female , Germ Cells/chemistry , Germ Cells/ultrastructure , Immunohistochemistry , Karyotyping , Male , Microscopy, Electron , Octamer Transcription Factor-3 , Stem Cells/chemistry , Stem Cells/cytology , Stem Cells/ultrastructure , Swine , Swine, Miniature
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