Materials and reagents

Biological materials

1. Donor corneas

1. Dulbecco’s modified Eagle medium (DMEM/F12) (Thermo Fisher Scientific, Gibco, catalog number: 12634-010)

2. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco, catalog number: 10010-023)

3. DMEM (1 g/L D-glucose, with GlutaMAX) (Thermo Fisher Scientific, Gibco, catalog number: 10567-014)

4. Ham’s F12 (Thermo Fisher Scientific, Gibco, catalog number: 31765-035)

5. BioWhittaker antibiotic-antimycotic (100×) (Lonza, catalog number: 17-745E)

6. Insulin-transferrin-selenium (100×) (Thermo Fisher Scientific, Gibco, catalog number: 41400-045)

7. Flexbumin (25%) (BioSupply, catalog number: 00944-0493-01)

8. 2-Phospho-L-Ascorbate (Tocris Biosci, catalog number: 5778)

9. Recombinant human epidermal growth factor (EGF) (Thermo Fisher Scientific, Gibco, catalog number: PHG6045)

10. Recombinant human platelet-derived growth factor-BB (PDGF-BB) (R&D, catalog number: 220-GMP-050)

11. Dexamethasone (Dex) (USP micronized) (Spectrum, catalog number: DE121)

12. Human serum (Innovative Res, catalog number: ISER-36670)

13. Collagenase (Collagenase NB6) powder (Nordmark, catalog number: N0002779)

14. Fibronectin coating substrate (MedChemExpress, catalog number: HY-P70593G)

15. TrypLE Select CTS (Thermo Fisher Scientific, Gibco, catalog number: A12859-01)

16. CryoStor CS10 (serum-free DMSO 10%) (BioLife Solutions, catalog number: 210373)

17. Trypan blue (0.4%) (Invitrogen, catalog number: T10282)

18. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H9892)

1. Recombinant human EGF (see Recipes)

2. Recombinant human PDGF (see Recipes)

3. Dexamethasone (Dex) solution (see Recipes)

4. Collagenase NB6 solution (see Recipes)

5. GMP fibronectin (see Recipes)

6. CryoStor CS5 (see Recipes)

7. GMP stem cell culture medium (see Recipes)

1. Recombinant human EGF

   Store the lyophilized EGF at -20 °C under desiccation until reconstitution. Prior to opening, briefly centrifuge the vial of lyophilized EGF at 400× g for 10–15 s to bring the contents to the bottom. Reconstitute EGF powders (500 mg) with sterile distilled water (1 mL volume) to a concentration of 0.5 mg/mL. Further dilute to 10 mg/mL with sterile DMEM/F12 added with 0.1% Flexbumin (carrier protein), aliquot at 0.5 mL, and store at -80 °C until use or at 2–8 °C for one month.

   Reagent	Final concentration	Quantity
   Recombinant human EGF	10 mg/mL	100 mL from 0.5 mg/mL stock
   DMEM/F12	1×	5 mL
   Flexbumin	0.1% (v/v)	20 mL from 25% stock




2. Recombinant human PDGF

   Store the lyophilized PDGF-BB at -20 °C under desiccation until reconstitution. Prior to opening, briefly centrifuge the vial of lyophilized PDGF-BB at 400× g for 10–15 s to bring the contents to the bottom. Reconstitute PDGF-BB powders (50 mg) with sterile hydrochloric acid (HCl, 4 mM) with 0.1% Flexbumin (carrier protein) to a concentration of 10 mg/mL, aliquot at 0.5 mL, and store at -80 °C until use or at 2–8 °C for one month.

   Reagent	Final concentration	Quantity
   Recombinant human PDGF-BB	10 mg/mL	One vial of 50 mg
   HCl	4 mM	5 mL
   Flexbumin	0.1% (v/v)	20 mL from 25% stock




3. Dexamethasone (Dex) solution

   Store the lyophilized Dex at -20 °C under desiccation until reconstitution. Prior to chemical preparation, wear PPE and a suitable eye protection device as Dex has a risk of causing eye damage/irritation. Thaw Dex at room temperature for 10–20 min. Weigh approximately 50 mg of powder and calculate the amount of distilled water to make a stock concentration of 10 mM with reference to the molecular weight 392.47. Further dilute to 10 mM with sterile DMEM/F12, aliquot at 100 mL volume, and store at -80 °C until use or at 2–8 °C for one month.

   Reagent	Final concentration	Quantity
   Dexamethasone	10 mM	5 mL from 10 mM stock
   DMEM/F12	1×	5 mL




4. Collagenase NB6 solution

   Store the lyophilized collagenase powder at 2–8 °C under desiccated conditions. Dissolve 100 mg of powder with 10 mL of sterile PBS (cGMP) to make a stock concentration of 10 mg/mL. Aliquot the solution to 0.15 mL in sterile 2 mL Eppendorf tubes and store at -20 °C. Prior to tissue digestion, thaw the collagenase aliquot at room temperature for 10–15 min and add 1.35 mL of DMEM/F12 with 0.1% Flexbumin and 1× antibiotics/antimycotic to make a working concentration of 1 mg/mL.

   Reagent	Final concentration	Quantity
   Collagenase NB6 powder	1 mg/mL	0.15 mL of 10 mg/mL stock
   DMEM/F12	1×	1.35 mL
   Flexbumin	0.1% (v/v)	6 mL
   Antibiotics/antimycotic	1×	15 mL from 100× stock




5. GMP fibronectin

   Store lyophilized fibronectin at -20 °C under desiccation for a maximum of 2 years. Thaw the powder at room temperature for 10–20 min. Prior to opening, briefly centrifuge the vial at 400× g for 10–15 s to bring the contents to the bottom. Add 1 mL of injection water to 500 mg of fibronectin powder to make a stock concentration of 500 mg/mL, aliquot to 50 mL volume, and store at -80 °C for up to a year or at 2–8 °C for a week.

   
   

   Fibronectin coating: Dilute GMP fibronectin at 500 mg/mL stock with sterile PBS to 10 mg/mL working concentration. Apply 1 mL per TCF25 (tissue culture flask, 25 cm²) and coat at room temperature for 15–30 min. Remove the solution, and the surface is ready for cell seeding.

   Reagent	Final concentration	Quantity
   Fibronectin	10 mg/mL	20 mL of 500 mg/mL stock
   PBS	1×	1 mL




6. CryoStor CS5

   Store CryoStor CS10 at 2–8 °C. Prior to use for CSSC cryopreservation, dilute CS10 with GMP culture medium (1:1 v/v) to make a working CS5 solution with 5% DMSO and keep at room temperature for immediate cell storage or at 2–8 °C for one week. The step of cell freezing requires the use of a controlled temperature apparatus (e.g., Mr. Frosty^TM freezing container with isopropanol).

   Reagent	Final concentration	Quantity
   GMP culture medium	0.5×	0.5 mL
   CryoStor CS10	0.5×	0.5 mL




7. GMP stem cell culture medium (GMP SCCM)

   Thaw the frozen reagents (including recombinant human EGF, recombinant human PDGF-BB, and Dex) at room temperature for approximately 10–15 min or at 2–8 °C overnight. Mix the thawed solutions by gently inverting the Eppendorf vial a few times, followed by a brief spinning with a benchtop micro-centrifuge.

   To prepare SCCM with 500 mL volume, mix 300 mL of DMEM (1,000 mg/L D-glucose) and 200 mL of Ham’s F12. Add reagents with the required volume, filter-sterilize, and bring the culture medium to room temperature before use.

   Reagents	Final concentration	Volume
   DMEM (1 g/L D-glucose, with GlutaMAX)	60% (v/v)	290 mL
   Ham’s F12	40% (v/v)	190 mL
   BioWhittaker antibiotic-antimycotic (100×)	1×	5 mL
   Insulin-transferrin-selenium (100×)	0.5×	2.5 mL
   2-Phospho-L-Ascorbate (100 mM)	0.5 mM	2.5 mL
   Recombinant human EGF (10 mg/mL)	10 ng/mL	0.5 mL
   Recombinant human PDGF-BB (10 mg/mL)	10 ng/mL	0.5 mL
   Dexamethasone, USP micronized (10 mM)	10 nM	0.5 mL
   Flexbumin (25%)	0.1% (v/v)	2 mL
   Human serum (100%)	2% (v/v)	10 mL

   
   

   Notes:

   1. All reagents used in this GMP culture formulation adhere to the GMP standards. During the development of the GMP-compliant formulation, we tested different GMP substitutes for the research-grade chemicals/reagents that are used in the lab-based culture medium [19]. This was done to ensure that the formulation meets the GMP standard. The corresponding reagents were comprehensively evaluated against the original research-grade components through rigorous cellular and molecular assays using at least three primary donor CSSC batches. After evaluating multiple GMP-grade reagents, we meticulously developed an optimized formulation that closely mirrors the research-grade culture medium.

   2. It is worth noting that human serum can be lipid- and protein-rich, depending on the donors. This can cause the culture medium to appear cloudy with the presence of insolubilities, although it is sterile. For this reason, we suggest that the final medium should be filter-sterilized before use.

1. Cell strainer (40 and 70 μm pore size) (Fisher Scientific, catalog numbers: 22362537 and 22362548)

2. Cryovials (1.8 mL volume) (Fisher Scientific, catalog number: 1050026)

3. Tissue culture flask (TCF) 25 cm² (Corning, catalog number: 430168), 75 cm² (Genesse Scientific, GenClone, catalog number: 25-209); tissue culture dish (TCD) 30 mm (Falcon, catalog number: 353001), 60 mm (Falcon, catalog number: 353002), 100 mm (diameter) (Genesse Scientific, GenClone, catalog number: 25-202); tissue culture plate (TCP) 6-well (Fisher Scientific, catalog number: EB012927)

4. Sterile syringe (10 mL volume) (BD, catalog number: 305482)

5. Sterile centrifuge tubes (15 mL and 50 mL volume) (VWR, catalog numbers: 525-1069 and 525-1014)

6. Sterile microcentrifuge tubes (1.5 mL and 2 mL volume) (Fisher Scientific, catalog numbers: 05-408-129 and 02-681-321)

7. Sterile syringe filters (0.22 mm pore size) (Nalgene, catalog number: 726-2520)

Equipment

1. Biological safety cabinet (Class II type A2/B2) (Baker, model: SteriGARD III Advance)

2. Phase-contrast microscope with 4×, 10×, and 20× objectives (Invitrogen, model: EVOS XL Core)

3. Stereomicroscope for dissection (0.8 to 16×) (AMScope, model: SM-1 LED 1445)

4. Humidified CO₂ incubator (Thermo, model: HERAcell 150i)

5. Temperature-controlled benchtop centrifuge (Thermo, catalog number: 75004381)

6. Automated cell counter (Invitrogen, model: Countess^TM)

7. Colibri toothed forceps

8. Vannas curved scissors (6 mm blade) for corneal procedures

9. Scalpel handle #3

10. Sterile surgical blade #10 (Integra Miltex carbon steel sterile surgical blades, model: 4-110)

11. Germinator glass bead sterilizer (Fisher Scientific, model: Microbead Sterilizer B1305-FIS)

12. Rotator (set at 30 rounds per minute, rpm)

13. Mr. Frosty^TM freezing container (with isopropanol) (Nalgene Cryo freezing container, model: 5100-0001)

14. Liquid nitrogen cell storage (Thermo, model: Thermolyne CY509975)

15. Digital balance (Denver Inst Co., model: TR2102)

Procedure