A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model
Trascrizione
Begin by detaching the cell cultures of interest with trypsin, and resuspending each cell type in 10 milliliters of fresh medium. Plate each cell culture in a new 10-centimeter dish, and allow the feeder cells to adhere in the cell culture incubator. After 30 minutes, collect the floating iPSCs, and pass the cultures through individual 70-micron cell strainers to remove the cell clusters for counting.
Resuspend each culture at a 1.5 x 107 cells per milliliter concentration in cold PBS, filtering again as necessary. Then, place 4-to-6-week-old female C57BL6 mice one at a time in a small animal restrainer, and adoptively transfer 200 microliters of one type of cell per mouse into the tail vein of each animal.
Use a dial gauge caliper to measure the swelling of both knees to establish the baseline measurement. 10 days after the cell transfer, use a 1-milliliter syringe to inject the mice with 100 micrograms of methylated BSA emulsified in complete Freund's adjuvant at the base of the tail of each experimental animal.
17 days after the cell transfer, induce arthritis by intra-articular injection of 20 micrograms of methylated BSA in 10 microliters of PBS into the left knee joint and 20 micrograms of methylated BSA, and 100 micrograms of whole ovalbumin in 10 microliters of PBS into the right knee joint of each anesthetized adoptively transferred animal.
Seven days after inducing arthritis, measure the knees again for calculation of the percent increase in the knee diameter and surgically excise the patellas. Fix the joints in formalin. Then, after decalcification in EDTA, embed the knees in paraffin, and obtain 4-micron sections for histochemical staining and analysis.
