Method for cultivation of cartilage and spheroids thereof
The present invention relates to a method for propagating or enriching cartilage cells and providing spheroids thereof, wherein the spheroids are useful for an autologous chondrocyte implantation (ACI) product. Hence, the invention in particular relates to the production of spheroids from articular cartilage and use thereof.
1 . A method for propagating or enriching cartilage cells and providing spheroids thereof comprising the steps:
(a) obtaining cartilage cells from tissue of human or animal origin as starting material,
(b) cultivating the cartilage cells from step (a) as a monolayer (2D) on a substrate up to a first passage,
(b1) passaging and cultivating the cartilage cells from step (b) as a monolayer (2D) on a substrate,
(c) taking the cultivated cartilage cells from the monolayer from step (b1) and cultivating said cultivated cartilage cells in a suspension in a 3D environment to form spheroids, and
(d) collecting the spheroids,
wherein the total cultivation time of step (b), step (b1), and step (c) is equal to or less than 55 days, the time duration of step (b) is more than 10 days and equal to or less than 16 days, and the time duration of step (c) is from 15 days to less than 28 days.
2 . The method according to claim 1 , wherein the spheroids consist of more than 3,000 cells.
3 . The method according to claim 1 , wherein the spheroids consist of cells in the range of 3,000 to 200,000 cells.
4 . The method according to claim 1 , wherein the spheroids consist of cells in the range of 3,500 to 75,000 cells.
5 . The method according to claim 1 , wherein the spheroids have a diameter in the range of 100 to 1,000 μm.
6 . The method according to claim 1 , wherein the spheroids have a diameter in the range of 200 to 900 μm.
7 . The method according to claim 1 , wherein the spheroids have a diameter in the range of 240 to 870 μm.
8 . The method according to claim 1 , wherein the spheroids, during use for the treatment of patients having cartilage defects or bone defects, display a reduced number of non-responders or therapeutic failures among the treated patients, as compared to spheroids produced by a method in which the total cultivation time of step (b), step (b1), and step (c) is greater than 55 days, the time duration of step (b) is 10 days or less or greater than 16 days, or the time duration of step (c) is less than 15 days or greater than or equal to 28 days.
9 . The method according to claim 8 , wherein the cartilage defects or bone defects are traumatic cartilage defects or traumatic bone defects or lesions.
10 . The method according to claim 8 , wherein the non-responders or therapeutic failures among the treated patients have a Knee injury and Osteoarthritis Outcome Score (KOOS) below 8.
11 . The method according to claim 1 , wherein the spheroids, during use for the treatment of patients having traumatic cartilage or bone lesions, cartilage degeneration, bone degeneration, osteoarthritis, or for therapeutic cartilage regeneration or bone regeneration in vivo, display a reduced number of non-responders or therapeutic failures among the treated patients, as compared to spheroids produced by a method in which the total cultivation time of step (b), step (b1), and step (c) is greater than 55 days, the time duration of step (b) is 10 days or less or greater than 16 days, or the time duration of step (c) is less than 15 days or greater than or equal to 28 days.