Experimental Study Of Separation And Purification Of Schwann Cells By Immunomagnetic Beads Method And Proliferation Of It

SCs, the glial cells of peripheral nervous system, are the main cell typewithin peripheral nerve stumps and play a crucial role in functional recovery ofinjured peripheral nerves. After peripheral nerve injury, SCs produce variousneurotrophic factors to promote and guide axon growth in the distal nerve stumps.So, we think SCs have a good prospect in recovery of peripheral nervous injuryand nerve tissue engineering. According to literatures, isolation and purificationof SCs for nerve engineering are difficult because of easy contamination fromfibroblasts, which proliferate much faster than SCs and become a predominantcell type over SCs during in vitro cultures.So the key point for in vivo transplantis how to get a great amount of highly purified and activated Schwann cells. Thepurpose of this experiment is to separate and purify Schwann cells byimmunomagnetic beads method, and find a optimal culture solution prescriptionto proliferate Schwann cells.Part one -- Separating and purifying Schwann cells by immunomagneticbeadsSD rats that had been born within 4 to 7 days were selected. Their bilateralsciatic nerves were dissected on germfree conditions. The nerve fascicles werethen extracted under 16×microscope. Two enzymes were used to digest the sciatic nerve specimens twice. Schwann cells were purified by immunomagnetic beadsafter being cultured in DF12 for 7 days, then passaged after two days culture.Schwann cell cultures were generated as confirmed by cytometry, energometryandimmunocytochemistry, drawing cell growth curve by MTT essay. Result: Thecells we obtained are Schwann cells, we can use immunomagnetic beads methodto separate and purify Schwann cells. The 96% vigor and 98% pure Schwann cellcultures were generated and passaged after two days. Conclusion: We can obtainmassive purified normal schwann cells to satisfy the need of tissue engineeredbioartificial nerve graft by this method.Part two-- Experiment study of proliferation of Schwann cellsTo digest the second filial generation Schwann cells to make cellsuspension, the density is 1×104/ml, to have an inoculation into 96-well cultureplate, every board has 6×12 holes, 200ul/hole.There are six groups in thisexperiment : Group A is the blank control group (primary culture solution);Group B: culture solution contains bFGF(50ng/ml); Group C: the culturesolution contains heparin sulfate proteoglycan protein(50ng/ml); Group D: theculture solution contains heparin sulfate proteoglycan protein (100ng/ml);Group E : the culture solution contains bFGF(50ng/ml) and heparin sulfateproteoglycan protein(50ng/ml); Group F: the culture solution contains bFGF(50ng/ml) and heparin sulfate proteoglycan protein(100ng/ml); cultured at37oC,5%CO2, to get one board at the same time every day and measure theabsorption factor (A value/OD value),to calculate the means of every group's Avalue and draw the cell growth curve, the time is the abscissa axis and A value isaxis of ordinate. Result: Groups B,C,D,E,F all are superior to the blank controlgroup, with statistics significance(P<0.05);there is on significant differencebetween Group B,C and Group D( P>0.05); Groups E,F are superior to group B,C,D(P<0.05);there is no significant difference between Group E and Group F(P>0.05).Conclusion:the culture solution which contains bFGF and HSPGcan make Schwann cells proliferate quickly; Group F is the best, Group E,F getthe top of cell division at the ninth day; Group A,B,C,D get the top at the seventhday.