Isolation, characterization and transplantation experiments of neural stem cells
Isolation, characterization and transplantation experiments of neural stem cells
Neural cells in the mammalian central nervous system (CNS) are derived from proliferating pluripotent stem cells or precursor cells that migrate, localize, and differentiate during development. However, the developmental mechanisms of neural cells in the CNS and the effects of environmental stimuli on them during development are still poorly understood. Similar to the hematopoietic system, a self-renewing stem cell population can give rise to a more restricted population of precursor cells during CNS development, but their existence cannot be confirmed because specific phenotypic markers for these precursor cells are not yet available. In the adult brain, only a small number of stem cells are present, and they differentiate at a slow rate at specific neurogenic sites into neural Woods
Operation method
Isolation, characterization and transplantation of neural stem cells
Principle
Specific parts of the embryonic or adult brain are dissected and removed, the tissue is digested using enzymatic digestion to dissociate the cells from the adherent tissues, and then other cell and tissue debris are removed by polyvinylpyrrolidone (Percoll) density gradient centrifugation (not required) to obtain partially purified stem cells that are inoculated (Figure 11-1). Move makings The anatomy of the hippocampal and spinal cord tissues of embryonic and adult rats is only briefly described here; a more detailed description of the experiments can be found in Chapter XIV. 1. Pregnant rats were deeply anesthetized by intraperitoneal injection of a combination of ketamine hydrochloride (44 mg/kg), acepr○mazine maleate (4?Omg/kg), and xylazine (romPun, 0.75 mg/kg). 2. The abdomen was incised, the uterine bicorns were removed and placed on ice, and the embryos were removed from the amniotic sac and placed in cold D>PBS. 3. the parietal-rump length of the embryo was measured to determine gestational age. 1. The embryonic brain was removed and placed in D-PBS. The fetal brain was fixed with forceps, the cortex was torn from the midline to one side and laid flat, the tissue was held with pointed forceps, and below the cortex, the hippocampus was sheared off with a curved ophthalmoscope. 2. Under the microscope, the hippocampal tissue was transferred into D-PBS. 3. remove the hippocampus from the contralateral side, strip the attached meningeal and vascular tissues with pointed forceps, and collect and mix the hippocampi of 15-20 embryos. 1. Under a microscope, with the embryo lying on its side in sterile I>PBS, the spinal cord was isolated at the cervical and lower lumbar segments by making a cut from the side of the spinal canal with ophthalmic scissors and making a similar incision on the contralateral side. 2. The spinal cord was removed and stripped of the surrounding attached tissues, and the spinal cords of 15 to 20 embryos were collected and mixed. 1. Adult (3-6 months) female Fisher344 rats were anesthetized, executed by decapitation, and the brain was removed and placed in a flat dish containing cold D-PBS. 2. The corpus callosum was severed under the microscope, the left and right cerebral cortices were separated above the thalamus, the posterior margins of the cerebral cortical hemispheres were turned up backward, and the fornix and hippocampal rut were separated along the medial and lateral margins of the hippocampus on both sides, and the hippocampus was removed. The rat was anesthetized, made to lie on its side, the spinal canal was incised from the side, an identical incision was made from the opposite side, the spinal cord was carefully removed, the connective tissue attached to the spinal cord was stripped, and the sacral, thoracic, lumbar, and cervical segments were dissected out under the microscope. The preparation of the animal to be transplanted is similar to the embryonic cell transplantation described in Chapter XIV. In addition, more detailed procedures for transplantation, perfusion of the animals, sectioning of the brain tissue, and identification of the transplanted cells by histochemical and immunocytochemical staining can be found in the experiments of Suhonen et al. (1997). These methods are briefly described below. 1. To facilitate sectioning on a frozen sectioning machine, the block of brain tissue containing the desired target area should be trimmed to the smallest possible size. 2. The trimmed brain tissue is embedded in OCT and fixed in the fixture of the frozen sectioning machine, and then frozen on dry ice for 15 min. OCT embedding helps the tissue to adhere to the frozen head. 3. Sections are cut and transferred to the wells of a tissue culture plate containing TCS (24 or 96 well plates), which can be stored in a -20 "C refrigerator for long periods of time. 1. Collect the culture medium containing neurospheres and transfer it to a 15 ml sterile centrifuge tube, centrifuge (1000 times, 3 min), and slowly aspirate the supernatant, taking care not to mix the cell sediment. 2. Resuspend the cells with Iml of EGF-containing serum-free N2 culture medium and gently blow the cells 10-20 times with a medium-bore pasteurized pipette to make a single-cell suspension. 3. Inoculate the cells into uncoated culture dishes or freeze them in liquid nitrogen for long-term storage. 1. Resuspend cells in serum-free N2 culture medium containing 1 ○% DMSO and appropriate growth factors. 2. Blow gently with a pipette to homogenize the cells and then dispense in sterile cryotubes at lml/tube. 3. Put the freezing tube upright into the foam box and place it in a 7010 refrigerator, so that the cells will cool down slowly during the freezing process. 4. The next day, transfer the cell freezing tube into a liquid nitrogen container. 1. Remove frozen cells from liquid nitrogen and immediately put them into a 37TC7JC bath and shake them from time to time to thaw them as soon as possible. 2. Dilute the thawed cells with DMEM:F12 culture medium and transfer to a 15 ml sterile centrifuge tube for centrifugation (lOOOg, 3 min), and aspirate the supernatant. 3. Wash the cells once with culture medium, and then resuspend the cells in Iml serum-free N2 culture medium, and gently suck and beat with a medium caliber pasteurized pipette to make a single-cell suspension. 4. Inoculate the rat-derived cells into PORN/Laminin-coated culture dishes; inoculate the mouse-derived cells into uncoated culture dishes with serum-free N2 culture medium containing appropriate growth factors. 1. Select relatively large (>100 cells/clone) and well-separated clones from the petri dish and mark them on the back of the dish. 2. Melt 3% agarose solution (agarose; prepared with PBS) in a microwave oven, and when it cools down to 45~50°C, mix 1 ml of agarose solution with 2 ml of ATV-trypsin, and keep it warm at 37°C. 3. Aspirate the culture solution from the petri dish, add the agarose/trypsin mixture immediately, shake the dish gently so that the liquid flows over the cell surface, and wait for 2~3 min for solidification. 4. Use a sterile pasteurized pipette to gently cut down along the periphery of each cell clone, pick out the agarose gel adhering to the cloned cells, and transfer them one by one to each well of a 24-well plate (the wells contain serum-free N2 culture medium with 50% conditioned culture medium and G418 added), and then, at the place of growth of the original cell clone, wash it with a small amount of (IOOul) culture medium twice, and then transfer the washings to the corresponding wells of the 24-well plate. 5. Replace the culture medium every 3~4d until the cultured cells reach the desired confluence, and prepare for passaging. 1. Collect the conditioned culture medium from the culture medium with high cell density (at least 24 hours incubation). 2. Centrifuge (1 000 g, 5 min) and freeze in small portions. To prevent contamination by residual cells, the conditioned culture can also be filtered. The properties of transplanted stem cells in vivo and their fate can be analyzed by immunocytochemical staining, in situ hybridization, and electron microscopic view^, etc., and only the immunocytochemical staining method is described here. ... 1.For detection of BrdU, CNS-derived tissue sections were floated in 5% formamide/2XSSC solution for pretreatment (65T:, 2 h), and then, the sections were incubated in 2mol/LHa for 30 min at 37℃. 2. The pretreated sections were analyzed by immunocytochemical staining as described above, and the expression of specific marker proteins could be observed by laser confocal or ^-light microscopy, and the co-localization of BrdU and specific marker proteins would be used to determine the fate of the transplanted cells in vivo. The number of cells in brain sections can be determined by unbiasedstereology, which has been described in detail (Sterio 1984; Petersonetal.1994;WestandSIomianka1998) and is only briefly described here. 1. The tissues containing the study subjects were serially sectioned and the samples were taken in the same, randomized way. According to the opticaldissectorprinciple all cells present in the tissue should have an equal chance of being sampled and counted. 2. Cells are counted directly on a three-dimensional unbiased counting grid according to the visual dissector sampling principle. 3. The overall cell count can be calculated directly from a known sample by theopiticalfractionatorprocedure, or by combining the density value obtained from the visual dissection count with the volume of the whole structure estimated by the Cavalieri step (Nv-VRef step). For more product details, please visit Aladdin Scientific website.
![通过P ercoll密度梯度离心，可以去除污染的组织碎片和其他细胞，从而使干细胞得 以部分纯化。 1•按9 份 Perooll, 1 份 10X:PBS的 比 例 （ V/V)，稀释Percoll储存液。 2 ■为获得不连续的PercoIl密度梯度，可在此基础上，制备实验所需的不同浓度 (50%、 4 0 % 、 3 0 % 、 2 0 % 和 1 0 % ) 的 Perooll梯度液，并将来源于酶消化和过滤后的细胞 悬液铺于 Perooll 梯 度 上 （Marie etal. 1997)。 3 . 室温条件下，离屯、 （400私， 20~30m in)。 4 . 收集分布于40% ~ 5 0 % P_ U梯度层之间的细胞。 5 . 将细胞用冷的PBS (含抗生素和两性霉素B) 稀释2〜5倍后离心（1〇〇〇 茗， 3min), 重复3 次。由于细胞沉淀较少，所以每次离心后，不应把上清液全部吸弃，而应留Iml左 右液体，以避免细胞丢失。 6 . 用 Iml培养液重悬细胞，取样计数，并以 个细胞/75on2 培养瓶]的密度接种细胞，如获得的细胞数较少，可将细胞接种于35mm 或 60mm的平皿中。 7 •通过Percoll连续密度梯度离心同样可分离纯化干细胞。将细胞与;Per00Il (1:1)混 合， i^ Li'后收集分布于上层（ 髓憐脂层， myelin Iayor) 和 底 层 （ 红细胞层）之间的液体。 以后的操作可重复步骤5 和 6。](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/B1468481624541pyuth2q43mpng_small.jpg)
