Isolated culture method for cartilage cells
A technology of separation culture and cell culture, applied in the field of cell culture, which can solve problems such as insufficiency of digestion, enzyme toxicity damage, difference in cell quantity and quality, and achieve the effect of maintaining vitality and improving efficiency
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Embodiment 1
[0027] Example 1: Isolation of Chondrocytes
[0028] 4-week-old New Zealand rabbits were sacrificed by ear vein injection, the articular cartilage of the hindlimb was separated under aseptic conditions, the fascia and perichondrium covering the cartilage tissue were peeled off, and put into a glass petri dish filled with PBS. The isolated cartilage tissue was cut into small pieces smaller than 1 mm.
[0029] The cartilage tissue was digested with 0.25% trypsin for 20 min; after washing with PBS for 3 times, it was digested with 0.05% type II collagenase for 18 h, and the cells were collected every 3 h.
[0030] Count the collected cells and inoculate them on 25cm 2 In cell culture flasks, add 1×10 5 chondrocytes in 5 mL of DMEM / F12 medium containing 10% FBS and 0.1 μg / mL EGF.
Embodiment 2
[0031] Example 2: Isolation of Chondrocytes
[0032] 4-week-old New Zealand rabbits were sacrificed by ear vein injection, the articular cartilage of the hindlimb was separated under aseptic conditions, the fascia and perichondrium covering the cartilage tissue were peeled off, and put into a glass petri dish filled with PBS. The isolated cartilage tissue was cut into small pieces smaller than 1 mm.
[0033] Add 0.25% trypsin to the cartilage tissue to digest for 30 minutes; after washing with PBS for 3 times, enzymatically digest with 0.05% type II collagenase for 18 hours, and collect cells every 3 hours.
[0034] Count the collected cells and inoculate them on 25cm 2 In cell culture flasks, add 1×10 5 chondrocytes in 5 mL of DMEM / F12 medium containing 10% FBS and 0.1 μg / mL EGF.
Embodiment 3
[0035] Example 3: Isolation of Chondrocytes
[0036] 4-week-old New Zealand rabbits were sacrificed by ear vein injection, the articular cartilage of the hindlimb was separated under aseptic conditions, the fascia and perichondrium covering the cartilage tissue were peeled off, and put into a glass petri dish filled with PBS. The isolated cartilage tissue was cut into small pieces smaller than 1 mm.
[0037] The cartilage tissue was digested with 0.25% trypsin for 20 min; after washing with PBS for 3 times, it was digested with 0.1% type II collagenase for 18 h, and the cells were collected every 3 h.
[0038] Count the collected cells and inoculate them on 25cm 2 In cell culture flasks, add 1×10 5 chondrocytes in 5 mL of DMEM / F12 medium containing 10% FBS and 0.1 μg / mL EGF.
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