Culture,differentiation And Identification Of Adult Autologous Neural Stem Cells From Abandoned Brain Tissues Of Patients During Craniotomy

Background and Objective Neural stem cells（NSCs）have the potential for self-renewal and multi-differentiation.The ability to proliferate,migrate,and differentiate into neurons,astrocytes,and oligodendrocytes in disease and injury,has become a hotspot in the study of neurological damage repair and regeneration.However,its clinical application is still difficult due to immune rejection and ethical limitations.With the development of cell biology technology,NSCs can be isolated from regions of fetal rats,adult rats and human embryos.Nevertheless,few reports have been published about NSCs obtained from adult brain tissue.This study intended to isolate and culture NSCs from abandoned brain tissues during neurosurgery in patients with craniocerebral surgery.In vitro,the autologous NSCs are expanded,cryopreserved,and the autologous NSCs library is established.This study will observe the stemness maintenance,lineage development and conduct differentiation studies of autologous NSC in vitro to provide an ethical autologous NSCs transplantation platform for patients with postoperative neurological deficits in brain injury and stroke.Methods（1）Modified primary culture of adult autologous NSCs:Approximately500mg of abandoned brain tissues were collected from 25 cases of craniocerebral operation.With a 10ml syringe,the tissue was pushed out into the dish and gently blowed to pieces about 10-500μm diameter with a dropper evenly.The supernatant liquid was discarded after centrifugation（1000 r/min,5 min）.Tissues and medium were inoculated at 1:40,5ml of complete medium（DMEM/F12,2%B27,20 ng/ml EGF,20ng/ml FGF,1%penicillin-streptomycin）was added.Finally,it was inoculated into a 25cm² culture flask and placed in 37℃5%CO ₂ incubator.（2）Subculture of adult autologous NSCs:The adherent growth of NSCs was induced with 4%FBS medium（DMEM/F12,2%FBS,20 ng/ml EGF,20 ng/ml FGF,1%penicillin-streptomycin）.After the cell proliferation reached a certain amount（about80%of the flask bottom）,the supernatant was discarded,and 2 ml of accutase was added for digestion for 5 min.The supernatant was centrifuged and removed,2%FBS culture solution was added,and the cell concentration was adjusted to 1×10⁹/L.Then the cell suspension was inoculated to a 25 cm² flask and placed in 37℃5%CO₂incubator.The adherent cells were digested and isolated every 2 to 3 days to prepare cell suspension.The subculture was performed with 1:2 or 1:3.（3）Induced differentiation of adult autologous NSCs:10%FBS medium（DMEM/F12,10%FBS,1%penicillin-streptomycin）was added to the NSCs suspension,and the cell concentration was adjusted to 1×10⁹/L.Then inoculated to a 25 cm² flask and placed in 37℃5%CO₂ incubator.The medium was replaced every 3 days and equal amount of new 10%FBS medium was added.（4）Identification of adult autologous NSCs and differentiated cells:Expression of Nestin（Nestin,marker for NSCs）,glial fibrillary acid protein（GFAP;marker for astrocytes）,β-Tubulin（marker for neurons）and SOX10（marker for oligodendrocytes）was detected by immunofluorescence and Western Blot（WB）.（5）Cryopreservation and recovery of adult autologous NSCs:The cell concentration was adjusted to 1 x 10⁹/L,then transferred to a cryotube and placed in a cryopreservation box at-80℃overnight for cryopreservation.The next day,it was placed in a liquid nitrogen tank and cryopreservated.During recovery,the cryotubes were quickly transferred to a 37℃water bath,and 5 ml of complete medium were added,and then the supernatant was discarded after centrifuged,5 ml of NSCs complete medium was added to resuspended again,then inoculated into a 25 cm² culture flask.the survival cells were observed and placed in 37℃5%CO₂ incubator.Results（1）Adult autologous NSCs primary culture:After 3 days of serum-free suspension culture,25 cases of brain tissue were collected.The suspended neurospheres with regular pattern were found under inverted microscope in 15 cases.After 5 days of culture,the volume of the neurospheres increased continuously.After 6-7 days,the neurospheres can be passaged.（2）The result of Nestin immunofluorescence of neurospheres:The neurospheres were Nestin positive by immunofluorescence with fluorescence microscope or confocal microscopy.（3）The outcomes of induced differentiation of adult autologous NSCs and identification of cells after induction:The neurospheres were inoculated in differentiation medium for1h and the cells were attached.After 3 days,the protuberances were found in a large number of cells.After 7 days,the immunofluorescence identification showed that neurospheres were differentiated intoβ-Tubulin-positive neuron-like cells,SOX10-positive oligodendrocyte-like cells and GFAP-positive astrocyte-like cells.（4）Cryopreservation and recovery of adult autologous NSCs:The NSCs were cryopreserved after passaged.Sampling recovery was performed on each passage,and found that the cells survived well and could be passaged again.At present,there are 66cryopreserved cells.The information were detail preserved such as cell source,culture algebra and cryopreservation date.Conclusion With modified primary culture,adult autologous neurospheres were successfully obtained from the abandoned brain tissue during open craniotomy,and the acquisition rate was 60%（15/25）.It can differentiate into neuron-like cells,oligodendrocyte-like cells and astrocyte-like cells after differentiation.After cryopreservation and rescovery,it still maintains its stem cell characteristics and can be extensively passaged and expanded.It provided an autologous neural stem cell transplantation platform for patients with neurological dysfunction after craniocerebral injury and stroke,which is both meets ethics and avoids immune rejection.