Simple separation and identification method of human placenta myofibroblasts

Abstract

The invention relates to a separation and identification method of human placenta myofibroblasts. The separation and identification method comprises the following steps of: mechanically crushing the decidual tissue of the placenta fetal surface, digesting the decidual tissue by DMEM digestive juice containing collagenase I and thereby separating the decidual tissue into individual karyocytes; cultivating the karyocytes by using a DMEM / F12 culture medium containing 10% of fetal calf serum under the conditions of 37 DEG C, 5% of CO2 and saturated humidity; inducing adherence to wall and completely replacing the liquid in 48 hours, removing cells not adhering to the wall, adding fresh culture medium, and after the cells adhering to the wall is mono-cloned, cultivating each monoclonal cell separately; and when the cells are fused by about 90%, performing pancreatin passage, thereby obtaining the myofibroblasts. The method further identifies the obtained myofibroblasts by the combined method of cell morphology, cell cycle, cell surface marker detection, cell adipogenesis and osteogenesis differentiative capacity detection, cell multipotential stem cell factor detection and cell desmin expression detection.

Description

field of invention [0001] The invention relates to a method for isolating myofibroblasts from the decidua surface of human placenta and identifying the myofibroblasts. Background technique [0002] The placenta is considered to be the most attractive "adult stem cell", which can differentiate into muscle cells, liver cells, osteoblasts, chondrocytes, adipocytes and other cells in a suitable in vivo or in vitro environment. Because the source of placental stem cells is relatively convenient, easy to separate and purify, it still has the characteristics of stem cells after multiple passages and expansion, and does not have the characteristics of immune rejection; therefore, it has become a hot spot in stem cell research in recent years. Transplantation, tissue engineering, genetic engineering and other fields have good application prospects. [0003] Myofibroblasts refer to muscle tissue precursor cells containing myofilaments in the cytoplasm, derived from mesoderm stem cell...

AI Technical Summary

Problems solved by technology

[0005] However, there are few reports on the separation of placental fibroblasts in China at present. Even if there are reports, they mainly focus on the separation of MSCs or stem cells, such as CN 102586184 A (Chinese Patent Application No. 201210044638.6, published on July 18, 2012 ) published an invention entitled "Method for Establishing Placental Mesenchymal Stem Cell Bank"; CN 101395266 A (Chinese Patent Application No. 200680053575.3, date of publication March 25, 2009)

[0006] In the international reports on placental fibroblasts, the isolation methods are all focused on enzyme digestion, but there are many enzymes involved in the digestion process: for example, when Zuzana Strakova of the University of Illinois in the United States isolated placental fibroblasts, collagenase, Deoxyribonuclease, hyaluronidase, and pronase are mixed with four enzymes to digest and obtain fibroblasts. The participation of multiple enzymes will inevitably lead to complex experimental procedures and unnecessary waste of resources.

Images