| Background:Mesenchymal stem cells(MSCs)are currently one of the most common stem cells in clinical practice.They were first discovered in the bone marrow and have the characteristics of stem cell self-renewal ability and multi-directional differentiation potential.Under specific inducing differentiation conditions,it can not only differentiate into osteocytes,chondrocytes,but also into adipocytes,and even neurons.Due to its many advantages such as easy isolation and expansion,immune regulation,and paracrine,MSCs could serve as the cell seeds and gene therapy vectors which hold a promising application prospect in cell therapy and tissue engineering.Existing research shows that MSCs can be successfully isolated from a variety of primary tissues,such as fat,pulp,placenta,and umbilical cord.However,bone marrow and fat-derived MSCs need to be collected invasively.There is a possibility of viral and bacterial contamination,and their ability to expand and differentiate will have greater heterogeneity due to donor age and physical health.In addition,although MSCs derived from fetal tissues showed strong proliferation and differentiation capabilities,controversy over the ethical issues limits its clinical application.Compared with other tissue sources,placenta is easier to obtain,medical waste is reused,pathogenic microorganism infection and immunogenicity are lower,and it is not subject to ethical restrictions.The placenta can be divided into four different levels:amniotic membrane(AM),chorionic membrane(CM),chorionic villi(CV),and decidual(DC).Various methods have been reported in the literature to isolate MSCs from human placenta and umbilical cord.However,they have not compared the advantages and disadvantages of these methods and the biological characteristics of isolated MSCs.At present,there is no optimal method for isolating placental mesenchymal stem cells at different levels.There are three commonly reported methods for isolating placental mesenchymal stem cells:tissue explant method,enzyme digestion method,and perfusion method.We first compared the yield and success rate and time of first passage of primary MSCs that could be obtained by isolating the same weight of tissue.In addition,we analyzed the biological characteristics of the five MSCs obtained by this method,and compared their surface markers,proliferation ability,and differentiation ability,so as to select placental mesenchymal stem cell seeds that are more suitable for clinical research and application.Object:In order to better evaluate the function of human placental mesenchymal stem cells obtained by different isolation methods,so as to provide clinically better placental-derived mesenchymal stem cell seedsMethods:We collected placenta from 35 healthy mothers and separated the placental tissue using three existing methods.Among them,30 cases had umbilical cord,amniotic membrane,chorionic mesenchymal layer,and chorionic villi and decidual membrane were subjected to two different culture methods:tissue explant method and enzymatic digestion method.The other 5 cases used umbilical vein perfusion method to isolate placental chorionic vascular mesenchymal stem cells.We first compared the yield and success rate and time of first passage of primary MSCSs that could be obtained by isolating the same weight of tissue,so we can obtained the best isolation method suitable for different levels of placenta.Secondly,we identified and analyzed the biological characteristics of surface markers,proliferation ability,and differentiation ability of 5 different tissue-derived MSCs that were successfully isolated.Results:MSCs can be obtained by all three methods:tissue explant method has the highest success rate of umbilical cord MSCs,reaching 96.67%,and the enzymatic digestion method has a success rate of more than 85%in extracting MSCs from five different tissues;tissue explant yield:UC>AM>CM>CV>DC,first passage time:UC<CM<CV<AM<DC,enzymatic digestion yield:AM>UC>CM>CV>DC,first passage time:CV<UC<CM<AM<DC,the difference is statistically significant(P<0.05);MSCs extracted by the three methods can meet the minimum standards set by the International Cell Therapy Association(ISCT)in 2006;umbilical cord and placenta are rich in a large number of mesenchymal stem cells in different tissues.And CD 105 was a membrane expression.Conclusions:All three culture methods can be used to isolate MSCs.The use of a handheld electric homogenizer to treat human umbilical cord and placental tissue saves time and effort,which has good tissue dispersion and red blood cell removal effects.It also can greatly improves the separation efficiency.2.The tissue explant method is most suitable for umbilical cord,and the enzyme digestion method can be applied to five kinds of tissues,especially the chorionic villi.3,The perfusion method is relatively complicated,and the uniformity of the isolated cells is relatively poor.4.Umbilical cord and placental chorionic mesenchymal stem cells have strong proliferative vigor,the most abundant sources,and high extraction efficiency.They are the ideal source for mass culture of primary mesenchymal stem cells for clinical treatment and research.5.MSCs from both sources have similar phenotypes and multi-directional differentiation potential,and fetal MSCs have stronger proliferation activity. |
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