Application Of Three-dimensional Simulated Microgravity Culture System On Adenohypophysis-derived Cell Culture

Background The pituitary gland is the most important endocrine gland.It is composed of adenohypophysis and neurohypophysis.Adenohypophysis is the center of human hormone secretion.The main cell types include Growth Hormone(GH)cells,Thyroid Stimulating Hormone(TSH)cells,Prolactin(PRL)cells,Gonadotropin(Gn H)cells,and Adrenocorticotropic hormone(ACTH)cells.All kinds of pituitary diseases,such as hypophysis dysplasia,tumor,infection,pituitary apoplexy,can cause hypophysis hormone secretion abnormality,affecting the normal growth and development of human body.Hypopituitarism is one of the most common diseases of the pituitary gland.Insufficient secretion of pituitary hormones can give rise to hypopituitarism.Its incidence rate is 1/4000 to 1/10000.At present,the way of clinical treatment for hypophysis is hormone replacement therapy.Patients need to receive life-long treatment with unstable effect and high cost.In recent years,cell therapy has made great progress in treating diabetes,heart failure,neuropathy and other fields.Cell therapy is expected to be a new treatment for hypophysis.All kinds of hormone secreting cells and pituitary stem cells(PSC)in adenohypophysis could be seed cells for the treatment of hypopituitarism.However,no systematic study on the isolation and culture of adenohypophysis hormone secreting cells and PSC in vitro has been reported.Rotating cell culture system(RCCS)is a three-dimensional culture system which can simulate microgravity on earth.Compared with two-dimensional culture,RCCS is more similar to the internal environment.It can avoid the ethical problems and the complexity of in vivo environment.RCCS has been widely used in the basic research of organ development,cell-cell interaction,cell differentiation and so on.At present,it has not been applied on adenohypophysis derived cells studies.Purpose In this study,the distribution of various cell types in mice pituitary gland was analyzed.PSC and adenohypophysis hormone secreting cells were isolated and cultured to elucidate their growth characteristics in vitro.Three-dimensional simulated microgravity culture system was used to further optimize the culture conditions,in order to provide fundamental knowledge of seed cells and culture system selection for cell transplantation in hypopituitarism treatment.Method 1.Mice were sacrificed by cervical dislocation,pituitary tissue was separated,paraffin embedded,tissue sectioned and H&E stained for histological observation.2.Adenohypophysis was isolated and digested with collagenase to obtain pituitary cells.3.Pituitary cells were cultured in vitro in DMEM/F12 medium containing 10% serum.Observing cell morphology and ELISA were used to determine hormone secretion.4.The pituitary cells were cultured in RCCS.The cell types were identified by immunofluorescence.ELISA were used to determine hormone secretion.5.Immunofluorescence was applied to label stem cell and pituitary cell markers,including SOX2,FSH,LH,ACTH,to analyze the distribution of different types of cells in the adenohypophysis.6.The PSC were isolated and cultured by enzyme digestion method in serum-free medium containing cytokines to study the in vitro sphere-forming ability of PSC.The expression of PSC markers was detected by immunofluorescence and QPCR.7.After differentiation culture,immunofluorescence and QPCR were used to detect the expression of various hormone cell related markers.The ratio of different cells was further calculated.8.In the RCCS,PSC were cultured for 14 days to form micro tissue,H&E staining was used to observe the structure.Immunofluorescence was used to observe the proliferation and differentiation of PSC.Result 1.The hypophysis consists of anterior lobe,posterior lobe and intermediate lobe.The adenohypophysis is mainly composed of anterior lobe and intermediate lobe.SOX2 positive cells were concentrated in the marginal area between the anterior and middle lobes of the pituitary,and scattered in the anterior lobe.All kinds of hormone secreting cells are scattered in anterior pituitary,including ACTH,FSH and LH hormone secreting cells.2.The primary pituitary cells were successfully isolated by mechanical isolation method and cultured in vitro by traditional cell culture method.Then the culture conditions were optimized by RCCS.The cell could be cultured for longer time,and the secretion of hormones was increased compared with the traditional culture method.3.PSC isolated from anterior pituitary can be propagated to the fourth generation in serum-free medium.The proportion of SOX2 positive cells in the first two generations of PSC can reach 97% and 85% respectively.The proportion of hormone secreting cells was low,among which ACTH positive cells were(14 ± 3)%,FSH positive cells were(0.3 ± 0.1)%,LH positive cells were(0.6 ± 0.2)%.By the third generation,the proportion of hormone secreting cells increased and the cell proliferation was slower.Among them,ACTH positive cells were(80 ± 8)%,FSH positive cells were(1 ± 0.2)%,LH positive cells were(2 ± 0.3)%,SOX2 positive cells were 50%.QPCR was used to detect the m RNA expression of PSC and hormone cell markers,and the results were consistent with immunofluorescence.4.PSC can proliferate and form a micro tissue by three-dimensional culture with RCCS.Some cells are still SOX2+ PSC,and some PSC can differentiate into GH+ and ACTH+ hormone secreting cells.Conclusion 1.There are PSC,adrenocorticotropic hormone cells,gonadotropin cells and other hormone secreting cells in adenohypophysis.2.All kinds of hormone secreting cells were successfully isolated from adenohypophysis and cultured in vitro.Although hormone secreting cells have secretory function,their proliferation ability is poor.RCCS can improve the survival ability and hormone secretion of adenohypophysis cells in vitro.3.PSC isolated from the anterior pituitary has self-renewal ability and multi-directional differentiation potential.As a result,PSC has the potential to become a seed cell for cell transplantation to treat hypopituitarism.In summary,compared with pituitary cells,PSC has better proliferation ability,which may represent a better seed cell for cell transplantation.RCCS is a better culture system.Therefore,application of PSC combined with RCCS on pituitary dysfunction treatment is of great potential for cell transplantation.PSC isolated from adenohypophysis has the ability of self-renewal and multi-directional differentiation,and can form adenohypophysis like tissue in RCCS system.In summary,this study explored the biological characteristics of adenohypophysis-derived cells in vitro,which laid a theoretical and experimental foundation for the related research of pituitary.At the same time,the results of this study also showed that RCCS is an ideal cell culture system in vitro.