Corneal endothelial cell (CEC) culturing methods and compositions

Abstract

Provided herein are methods and compositions for culturing corneal endothelial cells (CECs) to increase the number of cells that may be expanded from a single corneal donor. The resulting compositions can be used in CEC therapies to treat corneal endothelial diseases.

Description

Flask Type (Base Material): The base material is subject to change from passage to passage. In this Example, the following flasks were used for each passage and are also described in Fig. 1: Passage 0 (PO) was performed in Corning 6 well plate (CAT# 3506) or VWR 6 well plate (CAT#10861-696). Passage 1 (P1)_was performed in Corning T25 (CAT#430639) or Corning T75 (CAT#430641 U) or Corning 150 (CAT#430825) or Corning T25 CELLBIND (CAT#3289) or Corning T75 CELLBIND (CAT#3290) or Corning T150 CELL BIND (CAT#3291). Passage 2 (P2) was performed in Corning T25 (CAT#430639) or Corning T75 (CAT#430641 U) or Corning T150 (CAT#430825) or Corning T225 (CAT#431082) or Corning CellSTACK Culture Chamber, 1 chamber (CAT#3268) or Corning T25 CELLBIND (CAT#3289) or Corning T75 CELLBIND (CAT#3290) or Corning T150 CELL BIND (CAT#3291) or Corning T225 CELL BIND (CAT#3293) or Corning CellSTACK Culture Chamber CELLBIND, 1 chamber (CAT#3330). Passage 3 (P3) was performed in Corning T25 (CAT#430639) or Corning T75 (CAT#430641 U) or Corning T150 (CAT#430825) or Corning T225 (CAT#431082) or Corning CellSTACK Culture Chamber, 1 chamber (CAT#3268) or Corning CellSTACK Culture Chamber, 2 chamber (CAT#3269) or Corning CellSTACK Culture Chamber, 5 chamber (CAT#3313) Corning T25 CELLBIND (CAT#3289) or Corning T75 CELLBIND (CAT#3290) or Corning T150 CELL BIND (CAT#3291) or Corning T225 CELL BIND (CAT#3293) or Corning CellSTACK Culture Chamber CELLBIND, 1 chamber (CAT#3330) or Corning CellSTACK Culture Chamber CELLBIND, 2 chamber (CAT#3310) or Corning CellSTACK Culture Chamber CELL BIND, 5 chamber (CAT#3311). Passage 4 (P4) was performed in Corning T25 (CAT#430639) or Corning T75 (CAT#430641 U) or Corning T150 (CAT#430825) or Corning T225 (CAT#431082) or Corning CellSTACK Culture Chamber, 1 chamber (CAT#3268) or Corning CellSTACK Culture Chamber, 2 chamber (CAT#3269) or Corning CellSTACK Culture Chamber, 5 chamber (CAT#3313) or Corning CellSTACK Culture Chamber, 10 chamber (CAT#3271) or Corning T25 CELLBIND (CAT#3289) or Corning T75 CELLBIND (CAT#3290) or Corning T150 CELL BIND (CAT#3291) or Corning T225 CELL BIND (CAT#3293) or Corning CellSTACK Culture Chamber CELLBIND, 1 chamber (CAT#3330) or Corning CellSTACK Culture Chamber CELLBIND, 2 chamber (CAT#3310) or Corning CellSTACK Culture Chamber CELL BIND, 5 chamber (CAT#3311) or Corning CellSTACK Culture Chamber CELL BIND, 10 chamber (3312). Passage 5 (P5) was performed in Corning T25 (CAT#430639) or Corning T75 (CAT#430641 U) or Corning T150 (CAT#430825) or Corning T225 (CAT#431082) or Corning CellSTACK Culture Chamber, 1 chamber (CAT#3268) or Corning CellSTACK Culture Chamber, 2 chamber (CAT#3269) or Corning CellSTACK Culture Chamber, 5 chamber (CAT#3313) or Corning CellSTACK Culture Chamber, 10 chamber (CAT#3271) or Corning T25 CELLBIND (CAT#3289) or Corning T75 CELLBIND (CAT#3290) or Corning T150 CELL BIND (CAT#3291) or Corning T225 CELL BIND (CAT#3293) or Corning CellSTACK Culture Chamber CELLBIND, 1 chamber (CAT#3330) or Corning CellSTACK Culture Chamber CELLBIND, 2 chamber (CAT#3310) or Corning CellSTACK Culture Chamber CELL BIND, 5 chamber (CAT#3311) or Corning CellSTACK Culture Chamber CELL BIND, 10 chamber (3312). Culture Medium: L-ascorbic Acid Prep-. L-ascorbic acid was added to a phosphate buffer solution (PBS), which was then mixed, filtered, and stored at -20sC in cryovials. Y-27632 Prep\ PBS was added to Y-27632, which was then mixed, filtered, and stored at -20eC in cryovials. N-Media Prep-. Calcium chloride was added to CTS™ Opti-MEM™, which was then mixed and filtered. Separately, chondroitin sulfate was added to CTS™ Opti-MEM™, which was also mixed and filtered. Next, the calcium chloride solution, chondroitin sulfate solution, and fetal bovine serum were added to CTS™ Opti-MEM™, mixed and stored in a 2-8°C refrigerator. The fetal bovine serum concentration in the prepared media was 8% (v / v). NY-Media was then prepared by removing the N-Media from the refrigerator and warming it to room temperature. Aliquots of L-ascorbic Acid Prep and Y-27632 Prep were added to the N-Media Prep and thoroughly mixed to supplement the NY-Media. Methods Human functional human corneal cells were cultured starting at Passage 0 (PO) every 7 days, every 14 days, every 17 days, every 21 days, or every 30 days. The cell culture and expansion method described herein is outlined in Table 1 and in Table 2. Table 1: Overview of order of operations Unit Operation Receive and wash starting material Digestion and Wash PO Culture (35-45 days in culture) P1 Culture (30-45 days in culture) P2 Culture (30-45 days in culture) P3 Culture (21-45 days in culture) P4 Culture (21 days in culture) Harvest Formulate, Fill, and Finish Each passage (P0+) was performed at 37°C and a relative humidity of about 95 percent. The CO2 concentration, about 5 percent, was controlled to match physiologic conditions and to maintain a constant pH. The passages (PO +) were performed in in media with a pH of 7.2 to 7.5. O2 was not monitored or controlled. A PO of 7 days, 14 days, or 21 days in duration did not allow cell expansion comparable to the control process. It was determined that a PO of 35 to 45 days was necessary to enable expansion. A prolonged 35-45 day PO process (35-45 days) was assessed in conjunction with (i) 7 day passages for PI, P2, P3, and P4; (ii) 14 day passages for P1, P2, P3, and P4; or (iii) 21 day passages for P1, P2, P3, and P4. However, each of these expansion processes did not generate sufficient numbers of cells to provide -1000 doses per donor cornea. Finally, a prolonged 30-45 day passage for P1 and P2 (30-45 days) was assessed in conjunction with a prolonged passage 0 (35-45 days in length). Passages 3 and 4 were maintained at 21 days each. This process provided cell numbers that supported 1000 doses per donor at the end of passage 4. PO Culture: The OD (oculus dexter; right eye) tube and OS (oculus sinister; left eye) tube were removed from the incubator and centrifuged. The supernatant was then removed, NY-Media was added, and the wash was repeated. The cell starting material was resuspended and added to the first well in a 6-well plate. The cell starting material was mixed, as necessary, prior to adding additional cell starting material to the first well of the 6-well plate. The above procedure was repeated with the other cell starting material tube and added to the second well of the 6-well plate. The well plate was removed from the incubator with the spent media removed from each well and replaced with NY-Media. The spent media was saved for sterility testing. The well plate was then returned to the incubator. Passage took place when cells were in culture for at least 35 days, not to exceed 45 days. P1 Culture: NY-Media was added to the collection tube and the contents were mixed. The cell solution was then resuspended, dispensed into separate flasks, and the cells were evenly dispersed in the flasks. The flasks were placed in the incubator. The P1 flasks were removed from the incubator and the spent media was removed from the flasks. Fresh NY-Media was added back to the flasks and the flasks were returned to the incubator. This media change can be performed eleven additional times. The P1 Maintenance stage may take 30 days to 45 days. The NY-Media, PBS, and TrypLE (10x) were warmed to 37BC. The P1 flasks were removed from the incubator and inspected under a microscope. The spent media was sampled for endotoxins, gram stain, and mycoplasma testing. PBS was added to each flask and the flasks were agitated. The PBS was aspirated, additional PBS was added, and the flasks were placed in the incubator. After incubation, the flasks were removed, PBS was removed from each flask, and TrypLE (1 Ox) was added, and the flasks were incubated. The flasks were removed from the incubator and the flasks were tapped to aid in the disassociation of the cells from the flask. NY-Media was added to each flask, each flask was rinsed with the cell solution, and the cell solution from the flask was then added to the Collection Tube. The flask was then rinsed with NY-Media, and this rinse was added to the Collection Tube. The Collection Tube was centrifuged, and the supernatant was aspirated and discarded. The cell pellet was resuspended, and NY-Media was added to the Collection Tube. The contents in the Collection Tube were mixed, and a portion of the cell suspension was removed (termed Cell Suspension Aliquot_P1). Two aliquots were removed from Cell Suspension Aliquot_P1 and a cell count was performed on the two aliquots. After the completion of the cell count, the contents of the Cell Suspension Aliquot_P 1 were returned to the Collection Tube and the volume of the cell suspension recorded. P2 Culture: NY-Media was added to the Collection Tube and the contents were mixed. The cell solution was then resuspended, dispensed into separate flasks, and the cells were evenly dispersed in the flasks. The flasks were placed in the incubator. The P2 flasks were removed from the incubator and the spent media was removed from the flasks. Fresh NY-Media was added back to the flasks and the flasks were returned to the incubator. This media change may be performed eleven additional times. The P2 Maintenance stage may take 30 to 45 days. The NY-Media, PBS, and TrypLE (10x) were warmed to 37eC. The P1 flasks were removed from the incubator and inspected under a microscope. The spent media was sampled for endotoxins, gram stain, and mycoplasma testing. PBS was added to each flask and the flasks were agitated. The PBS is aspirated, additional PBS was added, and the flasks were placed in the incubator. After incubation, the flasks were removed, PBS was removed from each flask, and TrypLE (10x) was added, and the flasks were incubated. The flasks were removed from the incubator and the flasks were tapped to aid in the disassociation of the cells from the flask. NY-Media was added to each flask, each flask was rinsed with the cell solution, and the cell solution from the flask was then added to the Collection Tube. The flask was then rinsed with NY-Media, and this rinse was added to the Collection Tube. The Collection Tube was centrifuged, and the supernatant was aspirated and discarded. The cell pellet was resuspended, and NY-Media was added to the Collection Tube. The contents in the Collection Tube were mixed, and a portion of the cell suspension was removed (termed Cell Suspension Aliquot_P2). Two aliquots were removed from Cell Suspension Aliquot_P2 and a cell count was performed on the two aliquots. After the completion of the cell count, the contents of the Cell Suspension Aliquot_P2 were returned to the Collection Tube and the volume of the cell suspension recorded. P3 Culture: NY-Media was added to the Collection Tube and the contents were mixed. The cell solution was then resuspended, dispensed into separate flasks, and the cells were evenly dispersed in the flasks. The flasks were placed in the incubator. The P3 flasks were removed from the incubator and the spent media was removed from the flasks. Fresh NY-Media was added back to the flasks and the flasks were returned to the incubator. This media change may be performed eleven additional times. The P3 Maintenance may take 21 to 45 days. The NY-Media, PBS, and TrypLE (10x) were warmed to 37eC. The P1 flasks were removed from the incubator and inspected under a microscope. The spent media was sampled for endotoxins, gram stain, and mycoplasma testing. PBS was added to each flask and the flasks were agitated. The PBS was aspirated, additional PBS was added, and the flasks were placed in the incubator. After incubation, the flasks were removed, PBS was removed from each flask, and TrypLE (10x) was added, and the flasks were incubated. The flasks were removed from the incubator and the flasks were tapped to aid in the disassociation of the cells from the flask. NY-Media was added to each flask, each flask was rinsed with the cell solution, and the cell solution from the flask was then added to the Collection Tube. The flask was then rinsed with NY-Media, and this rinse was added to the Collection Tube. The Collection Tube was centrifuged, and the supernatant was aspirated and discarded. The cell pellet was resuspended, and NY-Media was added to the Collection Tube. The contents in the Collection Tube were mixed, and a portion of the cell suspension was removed (termed Cell Suspension Aliquot_P3). Two aliquots were removed from Cell Suspension Aliquot_P3 and a cell count was performed on the two aliquots. After the completion of the cell count, the contents of the Cell Suspension Aliquot_P3 were returned to the Collection Tube and the volume of the cell suspension recorded. P4 culture / harvest: After completion of the cell culture and expansion, the next steps of the methodology incorporate additional culturing (e.g., a P5 passage or later passage) and harvest steps. Table 2 provides a summary of the various passages and the time for each as described above. Table 2. Passage timeline Passage no. Time per passage (days) Total time (days) 0 35-45 35-45 1 30-45 65-90 2 30-45 950135 3 20-30 115-165 4 20-30 135-195 5 20-30 155-225 Results As shown in Fig. 2 and Table 3, the modified expansion process outlined above and in Fig. 1 and Table 2 resulted in a cell count that supports at least 1000 doses by P4 (see data enclosed by Circle “B” in Fig. 2). Table 3 summarizes the number of doses obtained in different cell culture formats at the indicated passages (Passage 3, Passage 4, or Passage 5) after passaging the cells in accordance with the timeline in Table 2. Table 3. Passage 3 Passage 4 Passage 5 Vessel # of vessels Doses # of vessels Doses # of vessels Doses T25 133 567 851 4204 6307 26575 T75 44 414 207 2670 1335 10816 T225 14 583 97 3698 616 11022 CS2 2 534 16 2276 68 5965 CS5 N / A N / A 6 2218 26 3971 T25: cell culture polystyrene flask; 25 cm2culture area (Thermo Scientific) T75: cell culture polystyrene flask; 75 cm2 culture area (Thermo Scientific) T225: cell culture polystyrene flask; 225 cm2 culture area (Thermo Scientific) CS2: Polystyrene CellSTACK; 2 chambers with 1272 cm2 cell growth area (Corning) CS5: Polystyrene CellSTACK; 5 chambers with 3180 cm2 cell growth area (Corning) As shown in Table 3, all culture formats assessed yielded cell counts that support over 1000 doses by P4. As cells continued to expand in P5, some culture vessels additionally provided the potential for larger scale cultures by P5 (e.g., >10,000 doses). These results suggest a reproducible process that can be adapted to different culture vessels. The foregoing studies also suggest that in order to obtain high numbers of cells (translating into 1000+ doses), the CECs should be kept in culture for about 30 to about 45 days at P0, P1, and P2. At a passage time of 30 to 45 days, cells go through a growth phase and then undergo contact inhibition, exit the growth phase and regain a non-proliferative state similar to their native state in vivo. So keeping the cells in culture for 30-45 days may help the cells transition from an in vivo non proliferative state (their native state in the cornea that was used as starting material) to in vitro culture conditions. Following P0-P2, P3 and P4 are shortened passages that keep the cells in an active growth state. This enables increased cell expansion scale. Another part of the process that increases cell expansion is to increase flask size progressively, i.e. PO: T25 > P1: T75 > P2: T150 > P3: T225 > P4: CS1 or CS2 or CS5. INCORPORATION BY REFERENCE The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

Claims

1. A method of culturing human corneal endothelial cells (CECs), said method comprising: culturing human corneal endothelial cells (CECs) in an initial passage 0 (PO); and expanding the human CECs through at least four additional passages comprising passage 1 (P1), passage 2 (P2), passage 3 (P3), and passage 4 (P4), wherein both P3 and P4 have time periods that are shorter than each of PO, P1, and P2;wherein CECs in each passage are cultured in a culture vessel; and wherein at least 1 x 107 cells are obtained by the end of P4.

2. The method of claim 1, wherein at least 1 x 108 cells are obtained by the end of P4.

3. The method of claim 2, wherein at least 1 x 109 cells are obtained by the end of P4.

4. The method of any one of claims 1 -3, further comprising expanding the CECs through passage 5 (P5).

5. The method of claim 4, wherein P5 is shorter in duration than each of PO, P1, and P2.

6. The method of claim 5, wherein at least 1 x 108 cells are obtained by the end of P5.

7. The method of claim 6, wherein at least 1 x 109 cells are obtained by the end of P5.

8. The method of claim 7, wherein at least 1 x 1010 cells are obtained by the end of P5.

9. The method of any one of the preceding claims, comprising culturing the CECs at PO for atleast 35 days.

10. The method of claim 9, comprising culturing the CECs at PO for 35 to 80 days.

11. The method of any one of the preceding claims, comprising expanding the CECs at P1 for at least 30 days.

12. The method of claim 11, comprising expanding the CECs at P1 for 30-80 days.

13. The method of any one of the preceding claims, comprising expanding the CECs at P2 for at least 30 days.

14. The method of claim 13, comprising expanding the CECs at P2 for 30-80 days.

15. The method of any one of the preceding claims, comprising expanding the CECs at P3 for at least 20 days.

16. The method of claim 15, comprising expanding the CECs at P3 for 20 to 45 days.

17. The method of claim 16, comprising expanding the CECs at P3 for 20 to 30 days.

18. The method of any one of the preceding claims, wherein P4 comprises expanding the CECs for at least 20 days.

19. The method of claim 18, wherein P4 comprises expanding the CECs at P4 for 20 to 30 days.

20. The method of any one of the preceding claims, wherein the culture vessel of the CECs in P3 and / or P4 is a stacked cell culture chamber comprising two or more layers.

21. The method of claim 20, wherein the stacked cell culture chamber comprises 2-10 layers.

22. The method of any one of the preceding claims, wherein the cell culture vessel has a medium volume of 130 to 8000 mL.

23. The method of claim 22, wherein the cell culture vessel has a medium volume of 130 to 2000 mL.

24. The method of any one of claims 1-21, wherein the cell culture vessel has a cell growth area of about 600 to about 6400 cm2.

25. The method of any one of claims 1-21, wherein the cell culture vessel has a cell growth area of about 1200 to about 6400 cm2.

26. A method of culturing corneal endothelial cells, the method comprising:culturing corneal endothelial cells (CECs) at passage 0 (P0) for at least 30 days;expanding the CECs at passage 1 (P1) for at least 30 days;expanding the CECs at passage 2 (P2) for at least 30 days;expanding the CECs at passage 3 (P3) for less than 30 days; andexpanding the CECs at passage 4 (P4) for less than 30 days.

27. A method of culturing corneal endothelial cells, the method comprising:culturing corneal endothelial cells (CECs) at passage 0 (P0) for 30-45 days;expanding the CECs at passage 1 (P1) for 30-45 days;expanding the CECs at passage 2 (P2) for 30-45 days;expanding the CECs at passage 3 (P3) for 20- 30 days; andexpanding the CECs at passage 4 (P4) for 20-30 days.

28. The method of claim 26 or 27, wherein at least 1 x 107 cells are obtained by the end of P4.

29. The method of claim 26 or 27, wherein at least 1 x 108 cells are obtained by the end of P4.

30. The method of claim 26 or 27, wherein the at least 1 x 109 cells are obtained by the end ofP4.

31. The method of any one of claims 26-30, further comprising expanding the CECs at passage 5 (P5) for less than 30 days.

32. The method of any one of claims 26-31, further comprising expanding the CECs at passage 5 (P5) for 20-30 days.

33. The method of claim 31 or 32, wherein at least 1 x 108 cells are obtained by the end of P5.

34. The method of claim 31 or 32, wherein at least 1 x 109 cells are obtained by the end of P5.

35. The method of claim 31 or 32, wherein at least 1 x 1010 cells are obtained by the end of P5.

36. The method of any one of claims 1-35, wherein each of the passages is performed at thesame temperature.

37. The method of any one of claims 1-36, wherein each passage is performed at a temperature of at least about 31°C.

38. The method of claim 37, wherein each passage is performed at a temperature of about 37°C.

39. The method of claim 37, wherein each passage is performed at a temperature of about 31 °C to 41 °C.

40. The method of any one of claims 1-39, wherein the CECs are derived from a single donor.

41. The method of any one of claims 1-40, wherein any one of PO, P1, P2, P3, or P4 are performed in a cell culture medium supplemented with ascorbic acid, fetal bovine serum, chondroitin sulfate, calcium chloride, and / or a Rho kinase inhibitor.

42. The method of claim 41, wherein the Rho kinase inhibitor is Y-27632.

43. The method of any one of claims 1-42, wherein at least 70% of the CECs are viable at the end of P4 as determined by a cell viability assay.

44. The method of claim 43, wherein at least 90% of the CECs are viable at the end of P4 as determined by a cell viability assay.

45. The method of any one of claims 1-44, wherein at least 70%, optionally at least 75%, of the CECs have a cell surface expression at the end of P4 of a marker selected from the group consisting of CD166 positive, CD44 negative to CD44 weakly positive, CD24 negative to weakly positive, CD44 negative to weakly positive, CD105 negative to weakly positive, CD26 negative to weakly positive, CD200 negative to weakly positive, and CD90 negative to weakly positive phenotypes.

46. The method of any one of claims 1-45, wherein at least 70%, optionally at least 75%, of the CECs have a cell surface expression at the end of P4 of a marker selected from the group consisting of sodium-potassium ATPase, ZO-1, VDAC3, SLC4A4, CLCN3, COL4A2, COL8A1, COL8A2, CDH2, CD98, CD166, CD340, Integrin a3pi, CD56, Prdx-6, CD248, SLC4A11, and CYYR1.

47. The method of any one of claims 1-46, wherein at the end of P4 at least 70% of the CECs are viable as determined by a cell viability assay;at least 75% of the CECs have a cell surface expression at the end of P4 of a marker selected from the group consisting of CD166 positive, CD44 negative to CD44 weakly positive, CD24 negative to weakly positive, CD44 negative to weakly positive, CD105 negative to weakly positive, CD26 negative to weakly positive, CD200 negative to weakly positive, and CD90 negative to weakly positive phenotypes; andat least 75% of the CECs have a cell surface expression at the end of P4 of a marker selected from the group consisting of sodium-potassium ATPase, ZO-1, VDAC3, SLC4A4, CLCN3, COL4A2, COL8A1, COL8A2, CDH2, CD98, CD166, CD340, Integrin a3pi, CD56, Prdx-6, CD248, SLC4A11, and CYYR1.

48. The method of any one of claims 1-47, wherein at least 70% of the CECs at the end of P4 have a hexagonal morphology as detected by microscopy.

49. The method of any one of claims 1-48, wherein the cells are grown on a cell culture surface comprising an extracellular matrix.

50. The method of any one of claims 1-49, wherein the cells are grown on a cell culture surface comprising an extracellular matrix protein.

51. The method of claim 50, wherein the extracellular matrix protein is selected from the group consisting of collagen, laminin, fibronectin, proteoglycan, SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, SWI / SNF-related matrix-associated actindependent regulator of chromatin subfamily B member 1, insulin-like growth factor-binding protein 5, vitronectin, fibrillin-1, fibrilin-2, tensin, Wnt-5b, citron Rho-interacting kinase, chondroitin sulphate proteoglycan 4, cyclin-dependent kinase 1, cyclin-dependent kinase 4, periostin, thrombospondin-4, Tubulin alpha chain-like 3, Tubulin alpha-1 B chain, Tubulin beta-1 chain, Tubulin beta-4A chain, and versican core protein.

52. The method of any one of claims 1-51, wherein the size of the culture vessel is increased with each passage.

53. The method of any one of claims 1-52, wherein the culture vessel is tissue-culture (TC)-treated.

54. The method of any one of claims 1-53, wherein the CECs are cultured for use in a therapeutic composition for use in humans.

55. A corneal endothelial cell (CEC) composition comprising CECs prepared by the method of any one of claims 1-54.

56. A pharmaceutical composition comprising the CEC composition of claim 55.

57. A tissue-culture treated (TC-treated) cell culture vessel comprising corneal endothelial cells (CECs).

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