Biology Reference
In-Depth Information
2.2.2. Seeding and Culture of Cartilage Constructs
1.
Mechanical punch: Obtain a unit that will punch polymer disks of less than or
equal to 10 mm in diameter.
2.
Petri dishes.
3.
Culture tubes (50 mL).
4.
Ethanol (100%).
5.
Culture medium D (construct culture medium): Prepare culture medium B and
add the following cell-culture grade components at the time of construct feeding:
insulin (50
µ
g/mL) and ascorbate (50
µ
g/mL). Culture medium without insulin
and ascorbate can be stored at 4
C for up to 2 wk. Insulin and ascorbate are added
at the time medium is added to cultures. Medium is replenished every 3-4 d.
°
6.
Culture dish:
Construct seeding step: 24-well (15-mm-diameter wells) nontissue culture-
treated dishes.
Construct culture step: 6-well (35-mm-diameter wells) tissue culture-treated dishes.
7.
Serological pipets (1 and 5 mL).
10
6
cells/mL culture medium D).
8.
Chondrocyte suspension (4-15
×
9.
Orbital, vibrating shaker: 300 rpm.
3. Methods
3.1. Tissue Harvest and Chondrocyte Isolation
Note:
Between 5-8
10
7
cells per gram of tissue are typically obtained from
skeletally immature animals.
1.
×
Skin femur, taking care not to puncture the joint capsule.
2.
Swab the joint with 70% isopropanol.
3.
Place joint and tissue harvesting materials in tissue-culture hood and procede
with the remaining steps in the hood.
4.
Make a medial parapateller incision in the distal femur to expose the articular
surface of the condyle and pateller groove. Collect tissue within the patellar
groove, patella, and condylar surfaces, being careful not to collect fibrous tissue,
which has a distinct texture and is usually more yellow in color than that of the
smooth, white, opaque cartilage. Dissect cartilage about 1 cm in length and 2-4 mm
in depth (depending upon the donor species and age), being careful not to collect
any calcified cartilage or into the subchondral bone.
5.
Place tissue chips in 50-mL tubes containing sterile culture medium A.
6.
After tissue dissection is completed, rinse once with culture medium A, then
transfer the tissue into freshly prepared culture medium C (10 mL per gram of tissue).
7.
Place closed tube(s), secured with parafilm, on their sides on an orbital shaker set
at 200-300 rpm and incubate overnight at 37
C.
8. Examine suspension to determine degree of digestion. Solution should be slightly
opaque with uniformly suspended cells.
9. Filter the cells through a cell filter (70
°
µ
m), discard trapped cells, and save
cell filtrate.
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