Experimental Study Of 3T3-L1 Preadipocytes Seeded Fibrin Glue For Adipose Tissue Engineering In Vitro

Objective:To culture 3T3-L1 preadipocytes in vitro and seeded fibrin glue, to study the adhesion, proliferation and differentiation of preadipocytes on fibrin glue. To explore the feasibility of fibrin glue for the scaffold materials in tissue engineering of preadipocytes. To provide an academic and practical foundation for the later research that fibrin glue used as a carrier for preadipocyte transplantation on prevention of epidural scar adhesion after laminectomy.Method:(1)The culture of 3T3-L1 preadipocytes in vitro: Cell line is cultivated in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37℃atmosphere of 5%CO2, change the medium on alternate days, transfer culture after fusion growth, proliferate to the number that required. (2) The experiment is consisted of two steps:1)Stage of preadipocyte culture; 2)Stage of induction and differentiation. The first stage is two groups, GroupA: Preadipocytes on fibrin glue; GroupB: Preadipocytes on pore plates. Digestive the well-grown cells with pancreatic enzyme(0.25%), count under the microscope, and make them into one-celled suspension, with a density of 1×105/ml. Dissolve Fibrinogen with DMEM medium supplemented with 10% FBS, and prepare the Fibrinogen solution with a density of 75mg/ml, dissolve Thrombin with Calcium Chloride solution with a density of 100u/ml. Take the one-celled suspension 100ul, Fibrinogen solution 200ul and Thrombin solution 100ul into 24 pore plates, mix them uniformity. 10 minutes standing in 37℃incubator, then make the preadipocytes fibrin glue complex, take another 100ul preadipocytes one-celled suspension into 24 pore plates to be the control group,the two groups are both 24 pores. Observe the adhesion,growth, proliferation of preadipocytes in two groups at different time point under the inverted phase contrast microscope. On day 1,3,5,7,9,11,13, observe the proliferation ability of cells in two groups by MTT assay. Randomly select 3 pores'cells in two groups, add 100ul MTT solution to the pores,4 hours standing in 37℃incubator, then inhale the supernatant,add DMSO 500ul to the pores,after10 minutes oscillation,have the crystals dissolved, take 150ul solution to 96 pore plates,measure the OD value at wavelength of 570nm, then Draw the cell growth curve. The second stage is 3 groups, Group C : cells induction on fibrin glue; Group D : cells induction on pore plates; Group E :cells isolated from fibrin glue induction on pore plates. Induction and differentiation are done in 24 pore plates, and every group is 18 pores,the number of cells in pores is 5×104. 5 days after cell inoculation add the inductor, the inductor is supplemented with IMBX(0.5mmol/L), insulin(5mg/L) Dexamethasone(1μmol/L). Observe the differentiation of preadipocytes in the three groups at different time point under the inverted phase contrast microscope. On day after induction 1,6,12, randomly select 5 pores'cells in three groups, use oil red O staining to measure the volume of adipose in preadipocytes. Fix the cells of C,D,E groups by isotonic buffer solution of 10% formaldehyde for 10 minutes, rinsed by PBS. Add oil red O into pores, after 15 minutes'standing rinse several times until clean. And then take the dyed pores plate into 37℃incubator to evaporate the moisture content in the pores plate, then add 1ml isopropanol, after 30 minutes shift-out the extraction dye solution by straw, measure the OD value at wavelength of 510nm on 722 spectrophotometer. The OD value means the volume of adipose in preadipocytes.Result: Under the reverse phase microscope, the preadipocytes adhere well on fibrin glue, and grew rapidly. The cell growth curve shows : the number of the cells on fibrin glue is higher than preadipocytes in pore plates on day 11(P<0.05). Preadipocytes on fibrin glue differentiated poorly, at 10 days after adding inducer , only a little preadipocytes on fibrin glue differentiate into adipocytes. The preadipocytes isolated from fibrin glue differentiate well as those can on pore plates.Conclusion: It is feasible to culture , proliferate and induce peadipocytes into mature adipocytes in vitro. Fibrin glue is very compatiblewith preadipocytes, it can be used as a carrier for preadipocytes, and it is expected scaffold materials for adipose tissue engineering. However it is necessary to research deeply the positive effect on preadipocytes differentiation and the differentiating potential of the preadipocytes on fibrin glue in vivo.