Preparation of CAR T cells in fibrin gel

Labprotocol                         Fibrin gel preparation                      Version 01

Created on: 09-Dec-2021

Created by: Ugi Uslu, June laboratory

Material:        TISSEEL [Fibrin Sealant]                       (Baxter, CAT: 1506078)

                       TBS (10X)                                             (Bio-Rad, CAT: 1706435)

                       Cell culture grade water                      (Corning, CAT: 25-055-CI)

                       Calcium chloride dihydrate                 (Sigma, CAT: C7902)

                       Human IL-7 Recombinant Protein       (Gibco, CAT: 200-07-1MG)

                       Human IL-15 Recombinant Protein     (Gibco, CAT: 200-15-500UG)

                       R10 cell culture media

Procedure (work sterile under the hood!):

- Store TISSEEL syringe (contains ready-to-use components of fibrinogen and thrombin) at -20 °C

- At day of experiment, put TISSEEL syringe at room temperature approximately 1-2 hours before use to allow enough time for thawing

- Preparation of fibrinogen solution:

• TISSEEL syringe contains 91mg/ml fibrinogen as per package insert
• Dillute 10X TBS with cell culture grade water at a 10:1 ratio to get 1X TBS
• Dillute fibrinogen component with 1X TBS to get desired end concentration of fibrinogen solution

- Preparation of thrombin solution:

• TISSEEL syringe contains 500U/ml thrombin as per package insert
• Dillute 10X TBS with cell culture grade water at a 10:1 ratio to get 1X TBS
• Dillute 1X TBS in 30nM calcium chloride
• Dillute syringe component with 1X TBS + 30nM CaCl₂ to get desired end concentration of thrombin solution

- Preparation of T cells:

• Thaw vial of frozen T cells approximately 2-3 hours before use and let them rest in cell culture media (R10) at 37°C
• Harvest and count T cells
• Prepare required amount of T cells in R10 media supplemented with 10ng/ml IL7 and 10ng/ml IL15
• Put T cells on ice

- Preparation of T cells in fibrin gel solution in animal facility:

• Put 37.5ul of prepared fibrinogen solution (in 1X PBS) in a new tube
• Add 25ul of T cells (in R10+IL7/15)
• Add 37.5ul of thrombin solution (in 1X PBS+CaCl₂)
• Total volume of T cell/fibrin gel solution: 100ul
       ➔ Use immediately for experiment since solution solidifies (time to solidification depends on concentration of thrombin solutions used)