| Background:The appearance of tissue engineering technology have provided a revolutionary routine for the problem of settling tissue defect thoroughly in clinic. However, the key of cellural component of tissue engineering is how to obtain the cells which have the characters of widely in origin, sufficient in quantity and well in quality.In recent years, the adipose derived stem cells (ASCs) from liposuction have become another hot spot after marrow stem cells. The mainly causes of why the adipose tissue is the origin of stem cells is that it has the advantages of easy to obtain stem cells from it, abundant sources, extracted repeatedly, harmless to health, cell proliferation rapidly and division in cycle but not easy senescence, and the discarding fluid portions during the liposuction is also the source of the stem cells, which have significant value in studying and using in developing research though lately found.Liposuction including fatty portion and fluid portion. It has been proved extensively that there are some stem cells called adipose derived stem cells (ASCs) in fat portion, which can be induced to different into osteoblast, cartilage, lipocytes, cardiac muscle cells, neuronal lineages, etc. The fluid portion of liposuction is made up of peripheral blood cell, adipocyte and adipose tissue fragment. However, there are many questions whether the liquid portion have the stem cells similar to the cells in fatty portion, or what is different in the cell quantity and quality between the stem cells extracted from liquid portion and from fatty portion? The research about liquid portion has no report in china.It is significant to recognize all of these questions so that we can solve the problem how to get more resource of seeds cell and culture efficiently. In our experiment we attempt to isolate and cultivated ASCs from the liquid portion of liposution and the activity of ASCs is evaluated. At the same time, the growth kinetics, morphology, cell senescence, surface marker profiles and differentiation capability of the ASCs derived from the fatty potion were compared to those derived from the liquid portion. It provides a new approach to study the ASCs and learn more data about the ASCs, which will be used in clinic in future.OBJECTIVE:1. To find a new method which can be used for isolation and cultivation of ASCs from liposuction, and the ASCs ability differentiated into lipocytes and osteoblast with the foundation of ASCs is the seed cell of adipose tissue engineering.2. To provide the laboratory evidence for ASCs to be used extensively in tissue engineering by charactering and comparing fatty portion derived and liquid portion derived ASCs' five characters including the growth kinetics, morphology, differentiation capability, cell senescence, surface marker profiles.METHODS:The liposuction aspirates was divided into fatty portion and liquid portion, ASCs were isolated and cultivated from each portion by collagenase digestion and directly centrifugate. The morphology and biology characters of the cells were observed in vitro. Cell activity were studied by MTT chromatometry ways and Cell growth curve was painted and the data obtained by that was statistically analyzed by computer with the software package SPSS 13.0 for windows. Cell cycle were detected by flow cytometry and cell senescence of ASCs was detected by studying the 3rd,4th,6th,8th generation cells' senescence selected stochastically during the culture through acridine orange staining ways. The cell surface markers were detected by flow cytometry and immunohistochemistry. Adipogenic and osteogenic lineage differentiations of the 4th generation of ASCs was assessed by Oil Red O and alizarin bordeaux staining respectively.RESULTS:There was a large amount of ASCs in the fatty portion and the liquid portion, including PLA cells and LAF cells. PLA cells were very similar to LAF cells in some characters such as cytomorphology, biology, cell senescence, cell surface markers, differentiations ability, etc, and they show identical in the cell quantity and quality.The statistical result indicated that the cell activity by MTT ways between PLA cells and LAF cells are very similar, the cells after two days' culture from being vaccinated had the highest activity. The cells cycle detected by flow cytometry showed that the 4th generation cells' differentiations ability between PLA cells and LAF cells were very similar and were all in the active condition. The result of acridine orange staining showed that the 3rd,4th,6th,8th, generation cells had no obviously senile signs. The majority of the 3rd,4th and 5th generation cells from PLA cells and LAF cells were CD44 and CD29 positive, CD34 were positive obviously in the 2nd generation cells and were little positive in the 4th and 5th generation cells, the cells of two portions have similar surface markers in mesenchymal stem cells surface markers and were CD29,CD44 positive, which were obtained by flow cytometry ways. vWF,CD31,CD105,SMA expressions were observed in ASCs by immunohistochemistry. Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks and significant fraction of the cells contained multiple, intracellular lipid-filled droplets that accumulated Oil Red-O. Afler 2 weeks' osteogenic induction, cells were positively stained by alizarin Bordeaux.CONCLUSION:A new process was set up to isolate ASCs by directly centrifugate and filtrate the fatty and the fluid portions of autologous liposuction aspirates of human without enzymatic digestion. The way of culture is convenient and easier to carry out. ASCs can be isolated from the liquid portion of liposuction aspirates and they show identical in the cell quantity and quality. LAF cells and PLA cells have similar characters in growth dynamics, morphology, cell senescence, surface marker profiles and differentiation ability, etc. Expressing of the cell surface marker of stem cells are also observed in ASCs and they can different into adipose and osteogenesis directionally. The results suggest that the ASCs, which were isolated with minimum limit artificial intervention, may be the ideal cells seeded in adipose tissue engineering in future.
|
PDF Full Text Request