Method for isolating and purifying schwann cells from adipose tissue
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A technique for Schwann cells and adipose tissue, applied in the field of cell biology, can solve the problems of high cost and low purification purity, and achieve the effect of promoting proliferation and low cost
Inactive Publication Date: 2011-09-21
SHANGHAI FIRST PEOPLES HOSPITAL
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The growth and reproduction speed of fibroblasts is much higher than that of Schwann cells. To remove fibroblasts before inoculation, there are many traditional purification methods for Schwann cells: the application of antibodies and anti-mitotic agents to inhibit the growth of fibroblasts, and the cells Toxicity may affect the proliferation of Schwann; tissue block differential attachment method purification purity is not high; serum-free culture method, the cost is high
The number of engineered Schwann cells should be at least 1×107 / ml to ensure that the neurotrophic factors and laminin produced by them can protect neurons and promote nerve regeneration. A large number of Schwann cells with relatively high purity can be obtained well, but clinical application needs to obtain a sufficient amount of cells for transplantation in the shortest possible time, so as to reduce the apoptosis of Schwann cells in vitro and the damage caused by denervation. occurrence of muscle atrophy
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Embodiment 1
[0049] Example 1 Separation and purification of Schwann cells
[0050] SD mice born at 6 days of dislocation were put to death in 75% alcohol and soaked for 5-10 minutes. Remove both groin fat under aseptic conditions.
[0051] 1. Take the fat of the mouse under aseptic conditions and cut it to 1mm with scissors 3 Size, place it in 3-10 times the amount of DMEM medium to wash, centrifuge and discard the supernatant.
[0052] 2. Digest the nerve fragments with 5-20 times the amount of tissue with a mass-volume ratio of 0.1-0.2% composite collagenase NB4 and 0.05-0.1% dispase mixed enzyme DMEM solution for 30-60 minutes.
[0053] 3. Pipette the suspension obtained in step 2 repeatedly with a pipette for 3 to 5 minutes.
[0054] 4. Centrifuge the suspension obtained in step 3 at 600g for 5-10 minutes, discard the supernatant, and obtain cell pellets.
[0055] 5. Resuspend the cells obtained in step 4 with Schwann cell culture medium (DMEM medium containing 2μM forskolin, 10ng / ml heregulin-...
Embodiment 3
[0064] Example 3 Comparison of digestion results of two digestive enzyme systems
[0065] A) Take primary cells
[0066] 2. 6 C57BL6 mice at 6 days old, after dislocation, put them in 75% alcohol and soak for 5-10 minutes
[0067] 3. Take out the inguinal fat tissue from both sides under aseptic condition and cut it to 1mm with scissors 3 Size, place it in 3-10 times the amount of DMEM medium to wash, centrifuge and discard the supernatant.
[0068] 4. Put the adipose tissue fragments into a centrifuge tube A containing 5ml 2% composite collagenase NB415ml and a centrifuge tube B containing 5ml 2% composite collagenase NB4 and 2ml 0.1% dispaseII respectively.
[0069] 5. Put it in an incubator containing 5% CO2 at 37°C, shake it every five minutes, and digest for about 1.5 hours.
[0070] 6. Centrifuge tube A and tube B at 600g for 5 minutes, collect cells, count and plant them in culture flasks, 0.5 minutes per flask.
[0071] 2) Purification of Schwann cells
[0072] After the primary ce...
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Abstract
The invention belongs to the field of cell biology, and relates to a method for isolating and purifying schwann cells from adipose tissue, and an application thereof. The method comprises the following steps: (1) under the aseptic condition, peeling the adipose tissue, then cutting the adipose tissue into pieces and cleaning; (2) digesting the adipose tissue pieces through a digestive enzyme solution whose volume is 3-20 times of the volume of the adipose tissue pieces, wherein the digestive enzyme solution is ferment enzyme, collagenase NB4 or a mixed liquor consisting of the ferment enzyme and the collagenase NB4; (3) repeated beating upon the resulting mixed suspension for 3-5 minutes through a pipette; (4) processing the dissociated cells through centrifuging to obtain a cell aggregate, wherein relative centrifugal force is 600-1500g, centrifuging time is 3-5 minutes; (5) resuspending the cells from the step (4) through a culture medium for schwann cells, and inoculating the resuspended cells on a culture dish coated with fibronectin. According to the present invention, purity of the schwann cells can approach 95%. With the method provided by present invention, a plurality of high-quality schwann cells with high purity can be obtained with low-cost and high-efficiency.
Description
Technical field [0001] The invention belongs to the field of cell biology, and relates to a method for separating and purifying Schwann cells from adipose tissue by using collagenase and enzyme enzyme and its application. Background technique [0002] Schwann cells (Schwann cells, SCs) are an important type of glial cells in the peripheral nervous system. They were first discovered by Schwann (1939) and play an important role in the myelination of myelinated nerve fibers and the repair of damaged nerves. It is also an important source of neurotrophic factors, which can secrete nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), ciliary nerve growth factor (CNTF), fibroblast growth factor (FGF), etc. The axons of adult mammals with central nerve injury lack the ability to regenerate, but after peripheral nerve injury, the ability to regenerate axons is relatively strong. Some scholars believe that this difference in axon regeneration ability is because the microen...
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