Processes for making a cellular fibronectin composition
The present application provides methods and processes for making and using a fibronectin composition, as well as methods for treating ocular conditions and/or disorders with the cellular fibronectin composition described herein.
1. A method of making a composition comprising mesenchymal stem cell (MSC)-derived fibronectin (FN), comprising:
(a) culturing mesenchymal stem cells (MSCs) in a cell culture medium, thereby generating conditioned medium that comprises factors comprising MSC-derived FN secreted by the MSCs;
(b) harvesting the conditioned medium thereby producing harvested conditioned medium; and
(c) filtering harvested conditioned medium to produce processed conditioned medium;
wherein the MSCs are bone marrow-derived.
2. The method according to claim 1 , wherein step (a) comprises:
(i) culturing the MSCs in a first culture medium;
(ii) removing the first culture medium from step (i) from the MSCs;
(iii) washing the MSCs in step (ii); and
(iv) adding a second culture medium and culturing for about 1-5 days.
3. The method according to claim 2 , wherein the first and/or second culture medium is selected from the group consisting of: hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (Bovine Serum Albumin-free), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (BME—with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM—with or without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E—with Earle's sale base), Medium M199 (M199H—with Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (MEM-E—with Earle's salt base), Minimum Essential Medium Eagle (MEM-H—with Hank's salt base), Minimum Essential Medium Eagle (MEM-NAA with non-essential amino acids), medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, and MDBC 153.
4. The method according to claim 2 , wherein the first and/or second culture medium is MSC Media and/or MSC-XF.
5. The method according to claim 2 , wherein the first and/or second culture medium is a serum-free medium.
6. The method according to claim 2 , wherein the first and/or second culture medium is supplemented with serum.
7. The method according to claim 2 , wherein the first and/or second culture medium is supplemented with human platelet lysate.
8. The method according to claim 1 , wherein the MSCs of step (a) are cultured in a first medium that is a growth factor-containing medium prior to being cultured in second medium that is growth factor-free at the time of being added to the MSCs.
9. The method according to claim 1 , wherein the culturing step (a) comprises growing the MSCs under a low oxygen partial pressure environment to facilitate cell growth and/or promote cell health.
10. The method according to claim 9 , wherein in the culturing step (a) the oxygen is at 5%, 10%, 15%, 20%, or 25%.
11. The method according to claim 1 , wherein the filtering step (c) comprises using a 0.45 μm filter, a 0.22 μm filter, 0.8 μm filter, and 0.65 μm filter, a low protein binding polyvinylidene difluoride (PVDF) membrane, and/or a PES (polyethersulfone) membrane.
12. The method according to claim 1 , wherein the filtering step (c) comprises using cross-flow filtration.
13. The method according to claim 1 , wherein step (c) comprises:
(1) filtering the harvested conditioned medium from step (b) to remove cell particulate;
(2) concentrating the filtered conditioned medium; and
(3) buffer exchanging with a formulation buffer.
14. The method according to claim 13 , wherein the concentrating step (2) comprises using hollow fiber filters, tangential flow filtration systems, or centrifugation based size exclusion techniques.
15. The method according to claim 13 , wherein the concentrating step (2) comprises using a centrifugation based size exclusion technique, wherein centrifugation based size exclusion technique employs a 3 kDa MW cutoff, at least a 4 kDa MW cutoff, at least a 5 kDa MW cutoff, at least a 6 kDa MW cutoff, at least a 7 kDa MW cutoff, at least a 8 kDa MW cutoff, at least a 9 kDa MW cutoff, at least a 10 kDa MW cutoff, at least a 11 kDa MW cutoff, at least a 12 kDa MW cutoff, at least a 13 kDa MW cutoff, at least a 14 kDa MW cutoff, at least a 15 kDa MW cutoff, at least a 16 kDa MW cutoff, at least a 17 kDa MW cutoff, at least a 18 kDa MW cutoff, at least a 19 kDa MW cutoff, at least a 20 kDa MW cutoff, at least a 21 kDa MW cutoff, at least a 22 kDa MW cutoff, at least a 23 kDa MW cutoff, at least a 24 kDa MW cutoff, at least a 25 kDa MW cutoff, at least a 26 kDa MW cutoff, at least a 27 kDa MW cutoff, at least a 28 kDa MW cutoff, at least a 29 kDa MW cutoff, and/or at least a 30 kDa MW cutoff.
16. The method according to claim 13 , wherein in the concentrating step (2) the filtered conditioned medium is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold.
17. The method according to claim 13 , wherein the buffer exchanging step (3) comprises buffer exchanging with a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
18. The method according to claim 13 , wherein in the buffer exchanging step (3) the conditioned medium is diluted less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25%.
19. The method according to claim 13 , wherein in the buffer exchanging step (3) the conditioned medium is buffer exchanged after the concentration step such that less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% or about 0% of the culture medium components remain.
20. The method according to claim 1 , further comprising characterizing the composition comprising MSC-FN using one or more assays selected from the group consisting of: a transwell migration assay, a human corneal epithelial cell proliferation assays, an endothelial cell tube formation assay, an oxidative stress prevention assays, a blood compatibility assay for hemolysis and hemagglutination, a depletion assay, a protein array assay, an enzyme-linked immunosorbent assay (ELISA), a mass spectrometry assay, and an immunoblotting assay.